Test module with microfluidic device having dialysis device, loc and interconnecting cap

ABSTRACT

A test module for analyzing a sample fluid containing target molecules, the test module having an outer casing with a receptacle for receiving the sample fluid, and, a microfluidic device having: a dialysis device in fluid communication with the receptacle and configured to separate the target molecules from other constituents of the sample, a lab-on-a-chip (LOC) device for analyzing the target molecules, and, a cap overlaying the LOC device and the dialysis device for establishing fluid communication between the LOC device and the dialysis device.

FIELD OF THE INVENTION

The present invention relates to diagnostic devices that usemicrosystems technologies (MST). In particular, the invention relates tomicrofluidic and biochemical processing and analysis for moleculardiagnostics.

CO-PENDING APPLICATIONS

The following applications have been filed by the Applicant which relateto the present application:

GBS001US GBS002US GBS003US GBS005US GBS006US GSR001US GSR002US GAS001USGAS002US GAS003US GAS004US GAS006US GAS007US GAS008US GAS009US GAS010USGAS012US GAS013US GAS014US GAS015US GAS016US GAS017US GAS018US GAS019USGAS020US GAS021US GAS022US GAS023US GAS024US GAS025US GAS026US GAS027USGAS028US GAS030US GAS031US GAS032US GAS033US GAS034US GAS035US GAS036USGAS037US GAS038US GAS039US GAS040US GAS041US GAS042US GAS043US GAS044USGAS045US GAS046US GAS047US GAS048US GAS049US GAS050US GAS054US GAS055USGAS056US GAS057US GAS058US GAS059US GAS060US GAS061US GAS062US GAS063USGAS065US GAS066US GAS067US GAS068US GAS069US GAS070US GAS080US GAS081USGAS082US GAS083US GAS084US GAS085US GAS086US GAS087US GAS088US GAS089USGAS090US GAS091US GAS092US GAS093US GAS094US GAS095US GAS096US GAS097USGAS098US GAS099US GAS100US GAS101US GAS102US GAS103US GAS104US GAS105USGAS106US GAS108US GAS109US GAS110US GAS111US GAS112US GAS113US GAS114USGAS115US GAS117US GAS118US GAS119US GAS120US GAS121US GAS122US GAS123USGAS124US GAS125US GAS126US GAS127US GAS128US GAS129US GAS130US GAS131USGAS132US GAS133US GAS134US GAS135US GAS136US GAS137US GAS138US GAS139USGAS140US GAS141US GAS142US GAS143US GAS144US GAS146US GAS147US GRR001USGRR002US GRR003US GRR004US GRR005US GRR006US GRR007US GRR008US GRR009USGRR010US GVA001US GVA002US GVA004US GVA005US GVA006US GVA007US GVA008USGVA009US GVA010US GVA011US GVA012US GVA013US GVA014US GVA015US GVA016USGVA017US GVA018US GVA019US GVA020US GVA021US GVA022US GHU001US GHU002USGHU003US GHU004US GHU006US GHU007US GHU008US GWM001US GWM002US GDI001USGDI002US GDI003US GDI004US GDI005US GDI006US GDI007US GDI009US GDI010USGDI011US GDI013US GDI014US GDI015US GDI016US GDI017US GDI019US GDI023USGDI030US GDI039US GDI040US GDI041US GPC001US GPC002US GPC003US GPC004USGPC005US GPC006US GPC007US GPC008US GPC009US GPC010US GPC011US GPC012USGPC014US GPC017US GPC018US GPC019US GPC023US GPC027US GPC028US GPC029USGPC030US GPC031US GPC033US GPC034US GPC035US GPC036US GPC037US GPC038USGPC039US GPC040US GPC041US GPC042US GPC043US GLY001US GLY002US GLY003USGLY004US GLY005US GLY006US GIN001US GIN002US GIN003US GIN004US GIN005USGIN006US GIN007US GIN008US GMI001US GMI002US GMI005US GMI008US GLE001USGLE002US GLE003US GLE004US GLE005US GLE006US GLE007US GLE008US GLE009USGLE010US GLE011US GLE012US GLE013US GLE014US GLA001US GGA001US GGA003USGRE001US GRE002US GRE003US GRE004US GRE005US GRE006US GRE007US GCF001USGCF002US GCF003US GCF004US GCF005US GCF006US GCF007US GCF008US GCF009USGCF010US GCF011US GCF012US GCF013US GCF014US GCF015US GCF016US GCF020USGCF021US GCF022US GCF023US GCF024US GCF025US GCF027US GCF028US GCF029USGCF030US GCF031US GCF032US GCF033US GCF034US GCF035US GCF036US GCF037USGSA001US GSA002US GSE001US GSE002US GSE003US GSE004US GDA001US GDA002USGDA003US GDA004US GDA005US GDA006US GDA007US GPK001US GMO001US GMV001USGMV002US GMV003US GMV004US GRD001US GRD002US GRD003US GRD004US GPD001USGPD003US GPD004US GPD005US GPD006US GPD007US GPD008US GPD009US GPD010USGPD011US GPD012US GPD013US GPD014US GPD015US GPD016US GPD017US GAL001USGPA001US GPA003US GPA004US GPA005US GSS001US GSL001US GCA001US GCA002USGCA003US

The disclosures of these co-pending applications are incorporated hereinby reference. The above applications have been identified by theirfiling docket number, which will be substituted with the correspondingapplication number, once assigned.

BACKGROUND OF THE INVENTION

Molecular diagnostics has emerged as a field that offers the promise ofearly disease detection, potentially before symptoms have manifested.Molecular diagnostic testing is used to detect:

-   -   Inherited disorders    -   Acquired disorders    -   Infectious diseases    -   Genetic predisposition to health-related conditions.

With high accuracy and fast turnaround times, molecular diagnostic testshave the potential to reduce the occurrence of ineffective health careservices, enhance patient outcomes, improve disease management andindividualize patient care. Many of the techniques in moleculardiagnostics are based on the detection and identification of specificnucleic acids, both deoxyribonucleic acid (DNA) and ribonucleic acid(RNA), extracted and amplified from a biological specimen (such as bloodor saliva). The complementary nature of the nucleic acid bases allowsshort sequences of synthesized DNA (oligonucleotides) to bond(hybridize) to specific nucleic acid sequences for use in nucleic acidtests. If hybridization occurs, then the complementary sequence ispresent in the sample. This makes it possible, for example, to predictthe disease a person will contract in the future, determine the identityand virulence of an infectious pathogen, or determine the response aperson will have to a drug.

Nucleic Acid Based Molecular Diagnostic Test

A nucleic acid based test has four distinct steps:

1. Sample preparation

2. Nucleic acid extraction

3. Nucleic acid amplification (optional)

4. Detection

Many sample types are used for genetic analysis, such as blood, urine,sputum and tissue samples. The diagnostic test determines the type ofsample required as not all samples are representative of the diseaseprocess. These samples have a variety of constituents, but usually onlyone of these is of interest. For example, in blood, high concentrationsof erythrocytes can inhibit the detection of a pathogenic organism.Therefore a purification and/or concentration step at the beginning ofthe nucleic acid test is often required.

Blood is one of the more commonly sought sample types. It has threemajor constituents: leukocytes (white blood cells), erythrocytes (redblood cells) and thrombocytes (platelets). The thrombocytes facilitateclotting and remain active in vitro. To inhibit coagulation, thespecimen is mixed with an agent such as ethylenediaminetetraacetic acid(EDTA) prior to purification and concentration. Erythrocytes are usuallyremoved from the sample in order to concentrate the target cells. Inhumans, erythrocytes account for approximately 99% of the cellularmaterial but do not carry DNA as they have no nucleus. Furthermore,erythrocytes contain components such as haemoglobin that can interferewith the downstream nucleic acid amplification process (describedbelow). Removal of erythrocytes can be achieved by differentially lysingthe erythrocytes in a lysis solution, leaving remaining cellularmaterial intact which can then be separated from the sample usingcentrifugation. This provides a concentration of the target cells fromwhich the nucleic acids are extracted.

The exact protocol used to extract nucleic acids depends on the sampleand the diagnostic assay to be performed. For example, the protocol forextracting viral RNA will vary considerably from the protocol to extractgenomic DNA. However, extracting nucleic acids from target cells usuallyinvolves a cell lysis step followed by nucleic acid purification. Thecell lysis step disrupts the cell and nuclear membranes, releasing thegenetic material. This is often accomplished using a lysis detergent,such as sodium dodecyl sulfate, which also denatures the large amount ofproteins present in the cells.

The nucleic acids are then purified with an alcohol precipitation step,usually ice-cold ethanol or isopropanol, or via a solid phasepurification step, typically on a silica matrix in a column, resin or onparamagnetic beads in the presence of high concentrations of achaotropic salt, prior to washing and then elution in a low ionicstrength buffer. An optional step prior to nucleic acid precipitation isthe addition of a protease which digests the proteins in order tofurther purify the sample.

Other lysis methods include mechanical lysis via ultrasonic vibrationand thermal lysis where the sample is heated to 94° C. to disrupt cellmembranes.

The target DNA or RNA may be present in the extracted material in verysmall amounts, particularly if the target is of pathogenic origin.Nucleic acid amplification provides the ability to selectively amplify(that is, replicate) specific targets present in low concentrations todetectable levels.

The most commonly used nucleic acid amplification technique is thepolymerase chain reaction (PCR). PCR is well known in this field andcomprehensive description of this type of reaction is provided in E. vanPelt-Verkuil et al., Principles and Technical Aspects of PCRAmplification, Springer, 2008.

PCR is a powerful technique that amplifies a target DNA sequence againsta background of complex DNA. If RNA is to be amplified (by PCR), it mustbe first transcribed into cDNA (complementary DNA) using an enzymecalled reverse transcriptase. Afterwards, the resulting cDNA isamplified by PCR.

PCR is an exponential process that proceeds as long as the conditionsfor sustaining the reaction are acceptable. The components of thereaction are:

1. pair of primers—short single strands of DNA with around 10-30nucleotides complementary to the regions flanking the target sequence

2. DNA polymerase—a thermostable enzyme that synthesizes DNA

3. deoxyribonucleoside triphosphates (dNTPs)—provide the nucleotidesthat are incorporated into the newly synthesized DNA strand

4. buffer—to provide the optimal chemical environment for DNA synthesis

PCR typically involves placing these reactants in a small tube (˜10-50microlitres) containing the extracted nucleic acids. The tube is placedin a thermal cycler; an instrument that subjects the reaction to aseries of different temperatures for varying amounts of time. Thestandard protocol for each thermal cycle involves a denaturation phase,an annealing phase, and an extension phase. The extension phase issometimes referred to as the primer extension phase. In addition to suchthree-step protocols, two-step thermal protocols can be employed, inwhich the annealing and extension phases are combined. The denaturationphase typically involves raising the temperature of the reaction to90-95° C. to denature the DNA strands; in the annealing phase, thetemperature is lowered to ˜50-60° C. for the primers to anneal; and thenin the extension phase the temperature is raised to the optimal DNApolymerase activity temperature of 60-72° C. for primer extension. Thisprocess is repeated cyclically around 20-40 times, the end result beingthe creation of millions of copies of the target sequence between theprimers.

There are a number of variants to the standard PCR protocol such asmultiplex PCR, linker-primed PCR, direct PCR, tandem PCR, real-time PCRand reverse-transcriptase PCR, amongst others, which have been developedfor molecular diagnostics.

Multiplex PCR uses multiple primer sets within a single PCR mixture toproduce amplicons of varying sizes that are specific to different DNAsequences. By targeting multiple genes at once, additional informationmay be gained from a single test-run that otherwise would requireseveral experiments. Optimization of multiplex PCR is more difficultthough and requires selecting primers with similar annealingtemperatures, and amplicons with similar lengths and base composition toensure the amplification efficiency of each amplicon is equivalent.

Linker-primed PCR, also known as ligation adaptor PCR, is a method usedto enable nucleic acid amplification of essentially all DNA sequences ina complex DNA mixture without the need for target-specific primers. Themethod firstly involves digesting the target DNA population with asuitable restriction endonuclease (enzyme). Double-strandedoligonucleotide linkers (also called adaptors) with a suitableoverhanging end are then ligated to the ends of target DNA fragmentsusing a ligase enzyme. Nucleic acid amplification is subsequentlyperformed using oligonucleotide primers which are specific for thelinker sequences. In this way, all fragments of the DNA source which areflanked by linker oligonucleotides can be amplified.

Direct PCR describes a system whereby PCR is performed directly on asample without any, or with minimal, nucleic acid extraction. It haslong been accepted that PCR reactions are inhibited by the presence ofmany components of unpurified biological samples, such as the haemcomponent in blood. Traditionally, PCR has required extensivepurification of the target nucleic acid prior to preparation of thereaction mixture. With appropriate changes to the chemistry and sampleconcentration, however, it is possible to perform PCR with minimal DNApurification, or direct PCR. Adjustments to the PCR chemistry for directPCR include increased buffer strength, the use of polymerases which havehigh activity and processivity, and additives which chelate withpotential polymerase inhibitors.

Tandem PCR utilises two distinct rounds of nucleic acid amplification toincrease the probability that the correct amplicon is amplified. Oneform of tandem PCR is nested PCR in which two pairs of PCR primers areused to amplify a single locus in separate rounds of nucleic acidamplification. The first pair of primers hybridize to the nucleic acidsequence at regions external to the target nucleic acid sequence. Thesecond pair of primers (nested primers) used in the second round ofamplification bind within the first PCR product and produce a second PCRproduct containing the target nucleic acid, that will be shorter thanthe first one. The logic behind this strategy is that if the wrong locuswere amplified by mistake during the first round of nucleic acidamplification, the probability is very low that it would also beamplified a second time by a second pair of primers and thus ensuresspecificity.

Real-time PCR, or quantitative PCR, is used to measure the quantity of aPCR product in real time. By using a fluorophore-containing probe orfluorescent dyes along with a set of standards in the reaction, it ispossible to quantitate the starting amount of nucleic acid in thesample. This is particularly useful in molecular diagnostics wheretreatment options may differ depending on the pathogen load in thesample.

Reverse-transcriptase PCR (RT-PCR) is used to amplify DNA from RNA.Reverse transcriptase is an enzyme that reverse transcribes RNA intocomplementary DNA (cDNA), which is then amplified by PCR. RT-PCR iswidely used in expression profiling, to determine the expression of agene or to identify the sequence of an RNA transcript, includingtranscription start and termination sites. It is also used to amplifyRNA viruses such as human immunodeficiency virus or hepatitis C virus.

Isothermal amplification is another form of nucleic acid amplificationwhich does not rely on the thermal denaturation of the target DNA duringthe amplification reaction and hence does not require sophisticatedmachinery. Isothermal nucleic acid amplification methods can thereforebe carried out in primitive sites or operated easily outside of alaboratory environment. A number of isothermal nucleic acidamplification methods have been described, including Strand DisplacementAmplification, Transcription Mediated Amplification, Nucleic AcidSequence Based Amplification, Recombinase Polymerase Amplification,Rolling Circle Amplification, Ramification Amplification,Helicase-Dependent Isothermal DNA Amplification and Loop-MediatedIsothermal Amplification.

Isothermal nucleic acid amplification methods do not rely on thecontinuing heat denaturation of the template DNA to produce singlestranded molecules to serve as templates for further amplification, butinstead rely on alternative methods such as enzymatic nicking of DNAmolecules by specific restriction endonucleases, or the use of an enzymeto separate the DNA strands, at a constant temperature.

Strand Displacement Amplification (SDA) relies on the ability of certainrestriction enzymes to nick the unmodified strand of hemi-modified DNAand the ability of a 5′-3′ exonuclease-deficient polymerase to extendand displace the downstream strand. Exponential nucleic acidamplification is then achieved by coupling sense and antisense reactionsin which strand displacement from the sense reaction serves as atemplate for the antisense reaction. The use of nickase enzymes which donot cut DNA in the traditional manner but produce a nick on one of theDNA strands, such as N. Alw1, N. BstNB1 and Mly1, are useful in thisreaction. SDA has been improved by the use of a combination of aheat-stable restriction enzyme (Ava1) and heat-stable Exo-polymerase(Bst polymerase). This combination has been shown to increaseamplification efficiency of the reaction from 10⁸ fold amplification to10¹⁰ fold amplification so that it is possible using this technique toamplify unique single copy molecules.

Transcription Mediated Amplification (TMA) and Nucleic Acid SequenceBased Amplification (NASBA) use an RNA polymerase to copy RNA sequencesbut not corresponding genomic DNA. The technology uses two primers andtwo or three enzymes, RNA polymerase, reverse transcriptase andoptionally RNase H (if the reverse transcriptase does not have RNaseactivity). One primer contains a promoter sequence for RNA polymerase.In the first step of nucleic acid amplification, this primer hybridizesto the target ribosomal RNA (rRNA) at a defined site. Reversetranscriptase creates a DNA copy of the target rRNA by extension fromthe 3′ end of the promoter primer. The RNA in the resulting RNA:DNAduplex is degraded by the RNase activity of the reverse transcriptase ifpresent or the additional RNase H. Next, a second primer binds to theDNA copy. A new strand of DNA is synthesized from the end of this primerby reverse transcriptase, creating a double-stranded DNA molecule. RNApolymerase recognizes the promoter sequence in the DNA template andinitiates transcription. Each of the newly synthesized RNA ampliconsre-enters the process and serves as a template for a new round ofreplication.

In Recombinase Polymerase Amplification (RPA), the isothermalamplification of specific DNA fragments is achieved by the binding ofopposing oligonucleotide primers to template DNA and their extension bya DNA polymerase. Heat is not required to denature the double-strandedDNA (dsDNA) template. Instead, RPA employs recombinase-primer complexesto scan dsDNA and facilitate strand exchange at cognate sites. Theresulting structures are stabilised by single-stranded DNA bindingproteins interacting with the displaced template strand, thus preventingthe ejection of the primer by branch migration. Recombinase disassemblyleaves the 3′ end of the oligonucleotide accessible to a stranddisplacing DNA polymerase, such as the large fragment of Bacillussubtilis Pol I (Bsu), and primer extension ensues. Exponential nucleicacid amplification is accomplished by the cyclic repetition of thisprocess.

Helicase-dependent amplification (HDA) mimics the in vivo system in thatit uses a DNA helicase enzyme to generate single-stranded templates forprimer hybridization and subsequent primer extension by a DNApolymerase. In the first step of the HDA reaction, the helicase enzymetraverses along the target DNA, disrupting the hydrogen bonds linkingthe two strands which are then bound by single-stranded bindingproteins. Exposure of the single-stranded target region by the helicaseallows primers to anneal. The DNA polymerase then extends the 3′ ends ofeach primer using free deoxyribonucleoside triphosphates (dNTPs) toproduce two DNA replicates. The two replicated dsDNA strandsindependently enter the next cycle of HDA, resulting in exponentialnucleic acid amplification of the target sequence.

Other DNA-based isothermal techniques include Rolling CircleAmplification (RCA) in which a DNA polymerase extends a primercontinuously around a circular DNA template, generating a long DNAproduct that consists of many repeated copies of the circle. By the endof the reaction, the polymerase generates many thousands of copies ofthe circular template, with the chain of copies tethered to the originaltarget DNA. This allows for spatial resolution of target and rapidnucleic acid amplification of the signal. Up to 10¹² copies of templatecan be generated in 1 hour. Ramification amplification is a variation ofRCA and utilizes a closed circular probe (C-probe) or padlock probe anda DNA polymerase with a high processivity to exponentially amplify theC-probe under isothermal conditions.

Loop-mediated isothermal amplification (LAMP), offers high selectivityand employs a DNA polymerase and a set of four specially designedprimers that recognize a total of six distinct sequences on the targetDNA. An inner primer containing sequences of the sense and antisensestrands of the target DNA initiates LAMP. The following stranddisplacement DNA synthesis primed by an outer primer releases asingle-stranded DNA. This serves as template for DNA synthesis primed bythe second inner and outer primers that hybridize to the other end ofthe target, which produces a stem-loop DNA structure. In subsequent LAMPcycling one inner primer hybridizes to the loop on the product andinitiates displacement DNA synthesis, yielding the original stem-loopDNA and a new stem-loop DNA with a stem twice as long. The cyclingreaction continues with accumulation of 10⁹ copies of target in lessthan an hour. The final products are stem-loop DNAs with severalinverted repeats of the target and cauliflower-like structures withmultiple loops formed by annealing between alternately inverted repeatsof the target in the same strand.

After completion of the nucleic acid amplification, the amplifiedproduct must be analysed to determine whether the anticipated amplicon(the amplified quantity of target nucleic acids) was generated. Themethods of analyzing the product range from simply determining the sizeof the amplicon through gel electrophoresis, to identifying thenucleotide composition of the amplicon using DNA hybridization.

Gel electrophoresis is one of the simplest ways to check whether thenucleic acid amplification process generated the anticipated amplicon.Gel electrophoresis uses an electric field applied to a gel matrix toseparate DNA fragments. The negatively charged DNA fragments will movethrough the matrix at different rates, determined largely by their size.After the electrophoresis is complete, the fragments in the gel can bestained to make them visible. Ethidium bromide is a commonly used stainwhich fluoresces under UV light.

The size of the fragments is determined by comparison with a DNA sizemarker (a DNA ladder), which contains DNA fragments of known sizes, runon the gel alongside the amplicon. Because the oligonucleotide primersbind to specific sites flanking the target DNA, the size of theamplified product can be anticipated and detected as a band of knownsize on the gel. To be certain of the identity of the amplicon, or ifseveral amplicons have been generated, DNA probe hybridization to theamplicon is commonly employed.

DNA hybridization refers to the formation of double-stranded DNA bycomplementary base pairing. DNA hybridization for positiveidentification of a specific amplification product requires the use of aDNA probe around 20 nucleotides in length. If the probe has a sequencethat is complementary to the amplicon (target) DNA sequence,hybridization will occur under favourable conditions of temperature, pHand ionic concentration. If hybridization occurs, then the gene or DNAsequence of interest was present in the original sample.

Optical detection is the most common method to detect hybridization.Either the amplicons or the probes are labelled to emit light throughfluorescence or electrochemiluminescence. These processes differ in themeans of producing excited states of the light-producing moieties, butboth enable covalent labelling of nucleotide strands. Inelectrochemiluminescence (ECL), light is produced by luminophoremolecules or complexes upon stimulation with an electric current. Influorescence, it is illumination with excitation light which leads toemission.

Fluorescence is detected using an illumination source which providesexcitation light at a wavelength absorbed by the fluorescent molecule,and a detection unit. The detection unit comprises a photosensor (suchas a photomultiplier tube or charge-coupled device (CCD) array) todetect the emitted signal, and a mechanism (such as awavelength-selective filter) to prevent the excitation light from beingincluded in the photosensor output. The fluorescent molecules emitStokes-shifted light in response to the excitation light, and thisemitted light is collected by the detection unit. Stokes shift is thefrequency difference or wavelength difference between emitted light andabsorbed excitation light.

ECL emission is detected using a photosensor which is sensitive to theemission wavelength of the ECL species being employed. For example,transition metal-ligand complexes emit light at visible wavelengths, soconventional photodiodes and CCDs are employed as photosensors. Anadvantage of ECL is that, if ambient light is excluded, the ECL emissioncan be the only light present in the detection system, which improvessensitivity.

Microarrays allow for hundreds of thousands of DNA hybridizationexperiments to be performed simultaneously. Microarrays are powerfultools for molecular diagnostics with the potential to screen forthousands of genetic diseases or detect the presence of numerousinfectious pathogens in a single test. A microarray consists of manydifferent DNA probes immobilized as spots on a substrate. The target DNA(amplicon) is first labelled with a fluorescent or luminescent molecule(either during or after nucleic acid amplification) and then applied tothe array of probes. The microarray is incubated in a temperaturecontrolled, humid environment for a number of hours or days whilehybridization between the probe and amplicon takes place. Followingincubation, the microarray must be washed in a series of buffers toremove unbound strands. Once washed, the microarray surface is driedusing a stream of air (often nitrogen). The stringency of thehybridization and washes is critical. Insufficient stringency can resultin a high degree of nonspecific binding. Excessive stringency can leadto a failure of appropriate binding, which results in diminishedsensitivity. Hybridization is recognized by detecting light emissionfrom the labelled amplicons which have formed a hybrid withcomplementary probes.

Fluorescence from microarrays is detected using a microarray scannerwhich is generally a computer controlled inverted scanning fluorescenceconfocal microscope which typically uses a laser for excitation of thefluorescent dye and a photosensor (such as a photomultiplier tube orCCD) to detect the emitted signal. The fluorescent molecules emitStokes-shifted light (described above) which is collected by thedetection unit.

The emitted fluorescence must be collected, separated from theunabsorbed excitation wavelength, and transported to the detector. Inmicroarray scanners, a confocal arrangement is commonly used toeliminate out-of-focus information by means of a confocal pinholesituated at an image plane. This allows only the in-focus portion of thelight to be detected. Light from above and below the plane of focus ofthe object is prevented from entering the detector, thereby increasingthe signal to noise ratio. The detected fluorescent photons areconverted into electrical energy by the detector which is subsequentlyconverted to a digital signal. This digital signal translates to anumber representing the intensity of fluorescence from a given pixel.Each feature of the array is made up of one or more such pixels. Thefinal result of a scan is an image of the array surface. The exactsequence and position of every probe on the microarray is known, and sothe hybridized target sequences can be identified and analysedsimultaneously.

More information regarding fluorescent probes can be found at:http://www.premierbiosoft.com/tech_notes/FRET_probe.html andhttp://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbook/Technical-Notes-and-Product-Highlights/Fluorescence-Resonance-Energy-Transfer-FRET.html

Point-of-Care Molecular Diagnostics

Despite the advantages that molecular diagnostic tests offer, the growthof this type of testing in the clinical laboratory has been slower thanexpected and remains a minor part of the practice of laboratorymedicine. This is primarily due to the complexity and costs associatedwith nucleic acid testing compared with tests based on methods notinvolving nucleic acids. The widespread adaptation of moleculardiagnostics testing to the clinical setting is intimately tied to thedevelopment of instrumentation that significantly reduces the cost,provides a rapid and automated assay from start (specimen processing) tofinish (generating a result) and operates without major intervention bypersonnel.

A point-of-care technology serving the physician's office, the hospitalbedside or even consumer-based, at home, would offer many advantagesincluding:

-   -   rapid availability of results enabling immediate facilitation of        treatment and improved quality of care.    -   ability to obtain laboratory values from testing very small        samples.    -   reduced clinical workload.    -   reduced laboratory workload and improved office efficiency by        reducing administrative work.    -   improved cost per patient through reduced length of stay of        hospitalization, conclusion of outpatient consultation at the        first visit, and reduced handling, storing and shipping of        specimens.    -   facilitation of clinical management decisions such as infection        control and antibiotic use.

Lab-on-a-Chip (LOC) Based Molecular Diagnostics

Molecular diagnostic systems based on microfluidic technologies providethe means to automate and speed up molecular diagnostic assays. Thequicker detection times are primarily due to the extremely low volumesinvolved, automation, and the low-overhead inbuilt cascading of thediagnostic process steps within a microfluidic device. Volumes in thenanoliter and microliter scale also reduce reagent consumption and cost.Lab-on-a-chip (LOC) devices are a common form of microfluidic device.LOC devices have MST structures within a MST layer for fluid processingintegrated onto a single supporting substrate (usually silicon).Fabrication using the VLSI (very large scale integrated) lithographictechniques of the semiconductor industry keeps the unit cost of each LOCdevice very low. However, controlling fluid flow through the LOC device,adding reagents, controlling reaction conditions and so on necessitatebulky external plumbing and electronics. Connecting a LOC device tothese external devices effectively restricts the use of LOC devices formolecular diagnostics to the laboratory setting. The cost of theexternal equipment and complexity of its operation precludes LOC-basedmolecular diagnostics as a practical option for point-of-care settings.

In view of the above, there is a need for a molecular diagnostic systembased on a LOC device for use at point-of-care.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides a test module for analyzinga sample fluid containing target molecules, the test module comprising:

an outer casing with a receptacle for receiving the sample fluid; and,

a microfluidic device having:

a dialysis device in fluid communication with the receptacle andconfigured to separate the target molecules from other constituents ofthe sample;

a lab-on-a-chip (LOC) device for analyzing the target molecules; and,

a cap overlaying the LOC device and the dialysis device for establishingfluid communication between the LOC device and the dialysis device.

Preferably, the fluid sample is a sample of biological materialincluding cells of different sizes, and the dialysis device has at leasttwo channels with a plurality of apertures fluidically connecting thechannels, the plurality of apertures being sized to correspond to apredetermined threshold size of the cells in the fluid sample.

Preferably, the at least two channels and the plurality of apertures areconfigured such that the sample flows though the channels and theapertures under capillary action.

Preferably, the at least two channels includes a target channel and awaste channel, the target channel being connected to the cap forcapillary driven flow to the LOC device.

Preferably, the target molecules are target nucleic acid sequenceswithin cells in the sample fluid, and the LOC device has a nucleic acidamplification section for amplifying the target nucleic acid sequences.

Preferably, the target nucleic acid sequences are within the cellssmaller than the predetermined threshold.

Preferably, the LOC device has a hybridization section with an array ofprobes for hybridization with the target nucleic acid sequences to formprobe-target hybrids.

Preferably, the probes are configured to form probe-target hybrids withthe target nucleic acid sequences, the probe-target hybrids beingconfigured to emit photons of light in response to an excitationelectric current.

Preferably, the LOC device has CMOS circuitry for operative control ofthe PCR section, the CMOS circuitry having a photosensor for sensingphotons emitted by the probe-target hybrids.

Preferably, the hybridization section has an array of hybridizationchambers containing the probes for hybridization with the target nucleicacid sequences.

Preferably, the photosensor is an array of photodiodes positionedadjacent each of the hybridization chambers respectively.

Preferably, the CMOS circuitry has a digital memory for storing datarelating to the processing of the fluid, the data including the probedetails and location of each of the probes in the array of hybridizationchambers.

Preferably, the CMOS circuitry has at least one temperature sensor forsensing the temperature at the array of hybridization chambers.

Preferably, the LOC device has a heater controlled by the CMOS circuitryusing feedback from the temperature sensor for maintaining the probesand the target nucleic acid sequences at a hybridization temperature.

Preferably, the photodiodes are less than 1600 microns from thecorresponding hybridization chamber.

Preferably, the probes have an electrochemiluminescent (ECL) luminophorethat emits a photon when in an excited state.

Preferably, the hybridization chambers have electrodes for exciting theECL luminophores with electrical current.

Preferably, the ECL probes each have a luminophore and a quencherpositioned proximate the luminophore for quenching photons emitted bythe luminophore such that hybridization with one of the target nucleicacid sequences moves the quencher away from the luminophore such thatthe photons are not quenched.

Preferably, the CMOS circuitry has bond-pads for electrical connectionto an external device, and is configured to convert output from thephotodiodes into a signal indicative of the ECL probes that hybridizedwith the target nucleic acid sequences, and provide the signal to thebond-pads for transmission to the external device.

Preferably, the cap has at least one channel for fluid communicationbetween the dialysis device and the LOC device, and a plurality ofreservoirs for holding liquid reagents for addition to the sample.

The easily usable, mass-producible, inexpensive, and portable diagnostictest module accepts a biochemical sample, uses a dialysis chip forseparating the cells of different dimensions, and separately processesthe nucleic acid content of the cells separated based on theirdimensions. The dialysis chip functionality extract additionalinformation from the sample and increases the sensitivity,signal-to-noise ratio, and dynamic range of the assay system.

The microfluidic multichip assembly provides for higher modularity.Surface-micromachined chips in the assembly would each be much smallerand disproportionately cheaper that a monolithic chip capable ofproviding the total functionality of the assembly. Alternatively a largeyet cost-effective dialysis chip can provide a high level of dialysiscapacity providing for further increased sensitivity, signal-to-noiseratio, and dynamic range for the assay system.

Functionally optimal, less expensive fabrication process is utilized tofabricate each surface-micromachined chip constituent of themicrofluidic multichip assembly, ie, the bioMST-only dialysis chip skipsall of the CMOS process steps and uses an inexpensive glass wafer.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the present invention will now be described byway of example only with reference to the accompanying drawings, inwhich:

FIG. 1 shows a test module and test module reader configured forfluorescence detection;

FIG. 2 is a schematic overview of the electronic components in the testmodule configured for fluorescence detection;

FIG. 3 is a schematic overview of the electronic components in the testmodule reader; FIG. 4 is a schematic representation of the architectureof the LOC device;

FIG. 5 is a perspective of the LOC device;

FIG. 6 is a plan view of the LOC device with features and structuresfrom all layers superimposed on each other;

FIG. 7 is a plan view of the LOC device with the structures of the capshown in isolation;

FIG. 8 is a top perspective of the cap with internal channels andreservoirs shown in dotted line;

FIG. 9 is an exploded top perspective of the cap with internal channelsand reservoirs shown in dotted line;

FIG. 10 is a bottom perspective of the cap showing the configuration ofthe top channels;

FIG. 11 is a plan view of the LOC device showing the structures of theCMOS+MST device in isolation;

FIG. 12 is a schematic section view of the LOC device at the sampleinlet;

FIG. 13 is an enlarged view of Inset AA shown in FIG. 6;

FIG. 14 is an enlarged view of Inset AB shown in FIG. 6;

FIG. 15 is an enlarged view of Inset AE shown in FIG. 13;

FIG. 16 is a partial perspective illustrating the laminar structure ofthe LOC device within Inset AE;

FIG. 17 is a partial perspective illustrating the laminar structure ofthe LOC device within Inset AE;

FIG. 18 is a partial perspective illustrating the laminar structure ofthe LOC device within Inset AE;

FIG. 19 is a partial perspective illustrating the laminar structure ofthe LOC device within Inset AE;

FIG. 20 is a partial perspective illustrating the laminar structure ofthe LOC device within Inset AE;

FIG. 21 is a partial perspective illustrating the laminar structure ofthe LOC device within Inset AE;

FIG. 22 is schematic section view of the lysis reagent reservoir shownin FIG. 21;

FIG. 23 is a partial perspective illustrating the laminar structure ofthe LOC device within Inset AB;

FIG. 24 is a partial perspective illustrating the laminar structure ofthe LOC device within Inset AB;

FIG. 25 is a partial perspective illustrating the laminar structure ofthe LOC device within Inset AI;

FIG. 26 is a partial perspective illustrating the laminar structure ofthe LOC device within Inset AB;

FIG. 27 is a partial perspective illustrating the laminar structure ofthe LOC device within Inset AB;

FIG. 28 is a partial perspective illustrating the laminar structure ofthe LOC device within Inset AB;

FIG. 29 is a partial perspective illustrating the laminar structure ofthe LOC device within Inset AB;

FIG. 30 is a schematic section view of the amplification mix reservoirand the polymerase reservoir;

FIG. 31 show the features of a boiling-initiated valve in isolation;

FIG. 32 is a schematic section view of the boiling-initiated valve takenthrough line 33-33 shown in FIG. 31;

FIG. 33 is an enlarged view of Inset AF shown in FIG. 15;

FIG. 34 is a schematic section view of the upstream end of the dialysissection taken through line 35-35 shown in FIG. 33;

FIG. 35 is an enlarged view of Inset AC shown in FIG. 6;

FIG. 36 is a further enlarged view within Inset AC showing theamplification section;

FIG. 37 is a further enlarged view within Inset AC showing theamplification section;

FIG. 38 is a further enlarged view within Inset AC showing theamplification section;

FIG. 39 is a further enlarged view within Inset AK shown in FIG. 38;

FIG. 40 is a further enlarged view within Inset AC showing theamplification chamber;

FIG. 41 is a further enlarged view within Inset AC showing theamplification section;

FIG. 42 is a further enlarged view within Inset AC showing theamplification chamber;

FIG. 43 is a further enlarged view within Inset AL shown in FIG. 42;

FIG. 44 is a further enlarged view within Inset AC showing theamplification section;

FIG. 45 is a further enlarged view within Inset AM shown in FIG. 44;

FIG. 46 is a further enlarged view within Inset AC showing theamplification chamber;

FIG. 47 is a further enlarged view within Inset AN shown in FIG. 46;

FIG. 48 is a further enlarged view within Inset AC showing theamplification chamber;

FIG. 49 is a further enlarged view within Inset AC showing theamplification chamber;

FIG. 50 is a further enlarged view within Inset AC showing theamplification section;

FIG. 51 is a schematic section view of the amplification section;

FIG. 52 is an enlarged plan view of the hybridization section;

FIG. 53 is a further enlarged plan view of two hybridization chambers inisolation;

FIG. 54 is schematic section view of a single hybridization chamber;

FIG. 55 is an enlarged view of the humidifier illustrated in Inset AGshown in FIG. 6;

FIG. 56 is an enlarged view of Inset AD shown in FIG. 52;

FIG. 57 is an exploded perspective view of the LOC device within InsetAD;

FIG. 58 is an enlarged plan view of the humidity sensor shown in InsetAH of FIG. 6;

FIG. 59 is a schematic showing part of the photodiode array of the photosensor;

FIG. 60 is an enlarged view of the evaporator shown in Inset AP of FIG.55;

FIG. 61 is a diagram of linker-primed PCR;

FIG. 62 is a schematic representation of a test module with a lancet;

FIG. 63 is a diagrammatic representation of the architecture of LOCvariant VII;

FIG. 64 is a plan view of LOC variant VIII with features and structuresfrom all layers superimposed on each other;

FIG. 65 is an enlarged view of Inset CA shown in FIG. 64;

FIG. 66 is a partial perspective illustrating the laminar structure ofLOC variant VIII within Inset CA shown in FIG. 64;

FIG. 67 is an enlarged view of Inset CE shown in FIG. 65;

FIG. 68 is a diagrammatic representation of the architecture of LOCvariant VIII;

FIG. 69 is a schematic illustration of the architecture of LOC variantXIV;

FIG. 70 is a schematic illustration of the architecture of LOC variantXLI;

FIG. 71 is a schematic illustration of the architecture of LOC variantXLIII;

FIG. 72 is a schematic illustration of the architecture of LOC variantXLIV;

FIG. 73 is a schematic illustration of the architecture of LOC variantXLVII;

FIG. 74 is a diagrammatic representation of the architecture of LOCvariant XI;

FIG. 75 is a circuit diagram of the differential imager;

FIG. 76 illustrates the reactions occurring during anelectrochemiluminescence (ECL) process;

FIG. 77 schematically illustrates three different anode configurations;

FIG. 78 is a schematic partial cross-section of the anode and cathode inthe hybridization chamber;

FIG. 79 schematically illustrates an anode in a ring geometry around theperipheral edge of a photodiode;

FIG. 80 schematically illustrates an anode in a ring geometry within theperipheral edge of a photodiode;

FIG. 81 schematically illustrates an anode with a series of fingers toincrease the length of its lateral edges;

FIG. 82 schematically illustrates the use of a transparent anode tomaximise surface area coupling and ECL signal detection;

FIG. 83 schematically illustrates the use of an anode affixed to theroof of the hybridization chamber to maximise surface area coupling andECL signal detection;

FIG. 84 schematically illustrates an anode interdigitated with acathode;

FIG. 85 shows a test module and test module reader configured for usewith ECL detection;

FIG. 86 is a schematic overview of the electronic components in the testmodule configured for use with ECL detection;

FIG. 87 shows a test module and alternative test module readers;

FIG. 88 shows a test module and test module reader along with thehosting system housing various databases;

FIG. 89 is a plan view of LOC variant L showing all the featuressuperimposed on each other and showing the locations of Insets GA to GL;

FIG. 90 is an enlarged view of Inset GD shown in FIG. 89;

FIG. 91 is an enlarged view of Inset GG shown in FIG. 89;

FIG. 92 is an enlarged view of Inset GH shown in FIG. 89;

FIG. 93 is a diagram of an electrochemiluminescence resonance energytransfer probe in a closed configuration;

FIG. 94 is a diagram of an electrochemiluminescence resonance energytransfer probe in an open and hybridized configuration;

FIG. 95 is a diagram of a primer-linked, luminescent linear probe duringthe initial round of amplification;

FIG. 96 is a diagram of a primer-linked, luminescent linear probe duringa subsequent amplification cycle;

FIGS. 97A to 97F diagrammatically illustrate thermal cycling of aluminescent primer-linked stem-and-loop probe;

FIG. 98 schematically illustrates a negative control luminescent probein its stem-and-loop configuration;

FIG. 99 schematically illustrates the negative control luminescent probeof FIG. 98 in its open configuration;

FIG. 100 schematically illustrates a positive control luminescent probein its stem-and-loop configuration;

FIG. 101 schematically illustrates the positive control luminescentprobe of FIG. 100 in its open configuration;

FIG. 102 is a diagrammatic representation of the architecture of amicrofluidic device with dialysis device, LOC device, interconnectingcap and electrochemiluminescence (ECL) detection;

FIG. 103 is a perspective view of a microfluidic device with dialysisdevice, LOC device and interconnecting cap;

FIG. 104 is a plan view showing the features of the dialysis device ofthe microfluidic device and showing the location of Inset JA;

FIG. 105 is an enlarged view of Inset JA shown in FIG. 104;

FIG. 106 is a plan view showing the features of the LOC device of themicrofluidic device and showing the locations of Insets JB to JJ;

FIG. 107 is an enlarged view of Inset JB shown in FIG. 106;

FIG. 108 is an enlarged view of Inset JC shown in FIG. 106;

FIG. 109 is an enlarged view of Inset JD shown in FIG. 106;

FIG. 110 is an enlarged view of Inset JE shown in FIG. 106;

FIG. 111 is an enlarged view of Inset JF shown in FIG. 106;

FIG. 112 is an enlarged view of Inset JG shown in FIG. 106;

FIG. 113 is an enlarged view of Inset JH shown in FIG. 106;

FIG. 114 is an enlarged view of Inset JJ shown in FIG. 106;

FIG. 115 is an enlarged view of the hybridization chamber of LOC variantL;

FIG. 116 is an enlarged view of the hybridization chamber array of LOCvariant L showing the distribution of calibration chambers;

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Overview

This overview identifies the main components of a molecular diagnosticsystem that incorporates embodiments of the present invention.Comprehensive details of the system architecture and operation are setout later in the specification.

Referring to FIGS. 1, 2, 3, 85 and 86, the system has the following toplevel components:

Test modules 10 and 11 are the size of a typical USB memory key and verycheap to produce. Test modules 10 and 11 each contain a microfluidicdevice, typically in the form of a lab-on-a-chip (LOC) device 30preloaded with reagents and typically more than 1000 probes for themolecular diagnostic assay (see FIGS. 1 and 85). Test module 10schematically shown in FIG. 1 uses a fluorescence-based detectiontechnique to identify target molecules, while test module 11 in FIG. 85uses an electrochemiluminescence-based detection technique. The LOCdevice 30 has an integrated photosensor 44 for fluorescence orelectrochemiluminescence detection (described in detail below). Bothtest modules 10 and 11 use a standard Micro-USB plug 14 for power, dataand control, both have a printed circuit board (PCB) 57, and both haveexternal power supply capacitors 32 and an inductor 15. The test modules10 and 11 are both single-use only for mass production and distributionin sterile packaging ready for use.

The outer casing 13 has a macroreceptacle 24 for receiving thebiological sample and a removable sterile sealing tape 22, preferablywith a low tack adhesive, to cover the macroreceptacle prior to use. Amembrane seal 408 with a membrane guard 410 forms part of the outercasing 13 to reduce dehumidification within the test module whileproviding pressure relief from small air pressure fluctuations. Themembrane guard 410 protects the membrane seal 408 from damage.

Test module reader 12 powers the test module 10 or 11 via Micro-USB port16. The test module reader 12 can adopt many different forms and aselection of these are described later. The version of the reader 12shown in FIGS. 1, 3 and 85 is a smart phone embodiment. A block diagramof this reader 12 is shown in FIG. 3. Processor 42 runs applicationsoftware from program storage 43. The processor 42 also interfaces withthe display screen 18 and user interface (UI) touch screen 17 andbuttons 19, a cellular radio 21, wireless network connection 23, and asatellite navigation system 25. The cellular radio 21 and wirelessnetwork connection 23 are used for communications. Satellite navigationsystem 25 is used for updating epidemiological databases with locationdata. The location data can, alternatively, be entered manually via thetouch screen 17 or buttons 19. Data storage 27 holds genetic anddiagnostic information, test results, patient information, assay andprobe data for identifying each probe and its array position. Datastorage 27 and program storage 43 may be shared in a common memoryfacility. Application software installed on the test module reader 12provides analysis of results, along with additional test and diagnosticinformation.

To conduct a diagnostic test, the test module 10 (or test module 11) isinserted into the Micro-USB port 16 on the test module reader 12. Thesterile sealing tape 22 is peeled back and the biological sample (in aliquid form) is loaded into the sample macroreceptacle 24. Pressingstart button 20 initiates testing via the application software. Thesample flows into the LOC device 30 and the on-board assay extracts,incubates, amplifies and hybridizes the sample nucleic acids (thetarget) with presynthesized hybridization-responsive oligonucleotideprobes. In the case of test module 10 (which uses fluorescence-baseddetection), the probes are fluorescently labelled and the LED 26 housedin the casing 13 provides the necessary excitation light to inducefluorescence emission from the hybridized probes (see FIGS. 1 and 2). Intest module 11 (which uses electrochemiluminescence (ECL) detection),the LOC device 30 is loaded with ECL probes (discussed above) and theLED 26 is not necessary for generating the luminescent emission.Instead, electrodes 860 and 870 provide the excitation electricalcurrent (see FIG. 86). The emission (fluorescent or luminescent) isdetected using a photosensor 44 integrated into CMOS circuitry of eachLOC device. The detected signal is amplified and converted to a digitaloutput which is analyzed by the test module reader 12. The reader thendisplays the results.

The data may be saved locally and/or uploaded to a network servercontaining patient records. The test module 10 or 11 is removed from thetest module reader 12 and disposed of appropriately.

FIGS. 1, 3 and 85 show the test module reader 12 configured as a mobilephone/smart phone 28. In other forms, the test module reader is alaptop/notebook 101, a dedicated reader 103, an ebook reader 107, atablet computer 109 or desktop computer 105 for use in hospitals,private practices or laboratories (see FIG. 87). The reader caninterface with a range of additional applications such as patientrecords, billing, online databases and multi-user environments. It canalso be interfaced with a range of local or remote peripherals such asprinters and patient smart cards.

Referring to FIG. 88, the data generated by the test module 10 can beused to update, via the reader 12 and network 125, the epidemiologicaldatabases hosted on the hosting system for epidemiological data 111, thegenetic databases hosted on the hosting system for genetic data 113, theelectronic health records hosted on the hosting system for electronichealth records (EHR) 115, the electronic medical records hosted on thehosting system for electronic medical records (EMR) 121, and thepersonal health records hosted on the hosting system for personal healthrecords (PHR) 123. Conversely, the epidemiological data hosted on thehosting system for epidemiological data 111, the genetic data hosted onthe hosting system for genetic data 113, the electronic health recordshosted on the hosting system for electronic health records (EHR) 115,the electronic medical records hosted on the hosting system forelectronic medical records (EMR) 121, and the personal health recordshosted on the hosting system for personal health records (PHR) 123, canbe used to update, via network 125 and the reader 12, the digital memoryin the LOC 30 of the test module 10.

Referring back to FIGS. 1, 2, 85 and 86 the reader 12 uses battery powerin the mobile phone configuration. The mobile phone reader contains alltest and diagnostic information preloaded. Data can also be loaded orupdated via a number of wireless or contact interfaces to enablecommunications with peripheral devices, computers or online servers. AMicro-USB port 16 is provided for connection to a computer or mainspower supply for battery recharge.

FIG. 62 shows an embodiment of the test module 10 used for tests thatonly require a positive or negative result for a particular target, suchas testing whether a person is infected with, for example, H1N1Influenza A virus. Only a purpose built USB power/indicator-only module47 is adequate. No other reader or application software is necessary. Anindicator 45 on the USB power/indicator-only module 47 signals positiveor negative results. This configuration is well suited to massscreening.

Additional items supplied with the system may include a test tubecontaining reagents for pre-treatment of certain samples, along withspatula and lancet for sample collection. FIG. 62 shows an embodiment ofthe test module incorporating a spring-loaded, retractable lancet 390and lancet release button 392 for convenience. A satellite phone can beused in remote areas.

Test Module Electronics

FIGS. 2 and 86 are block diagrams of the electronic components in thetest modules 10 and 11, respectively. The CMOS circuitry integrated inthe LOC device 30 has a USB device driver 36, a controller 34, aUSB-compatible LED driver 29, clock 33, power conditioner 31, RAM 38 andprogram and data flash memory 40. These provide the control and memoryfor the entire test module 10 or 11 including the photosensor 44, thetemperature sensors 170, the liquid sensors 174, and the various heaters152, 154, 182, 234, together with associated drivers 37 and 39 andregisters 35 and 41. Only the LED 26 (in the case of test module 10),external power supply capacitors 32 and the Micro-USB plug 14 areexternal to the LOC device 30. The LOC devices 30 include bond-pads formaking connections to these external components. The RAM 38 and theprogram and data flash memory 40 have the application software and thediagnostic and test information (Flash/Secure storage, e.g. viaencryption) for over 1000 probes. In the case of test module 11configured for ECL detection, there is no LED 26 (see FIGS. 85 and 86).Data is encrypted by the LOC device 30 for secure storage and securecommunication with an external device. The LOC devices 30 are loadedwith electrochemiluminescent probes and the hybridization chambers eachhave a pair of ECL excitation electrodes 860 and 870.

Many types of test modules 10 are manufactured in a number of testforms, ready for off-the-shelf use. The differences between the testforms lie in the on board assay of reagents and probes.

Some examples of infectious diseases rapidly identified with this systeminclude:

-   -   Influenza—Influenza virus A, B, C, Isavirus, Thogotovirus    -   Pneumonia—respiratory syncytial virus (RSV), adenovirus,        metapneumovirus, Streptococcus pneumoniae, Staphylococcus aureus    -   Tuberculosis—Mycobacterium tuberculosis, bovis, africanum,        canetti, and microti    -   Plasmodium falciparum, Toxoplasma gondii and other protozoan        parasites    -   Typhoid—Salmonella enterica serovar typhi    -   Ebola virus    -   Human immunodeficiency virus (HIV)    -   Dengue Fever—Flavivirus    -   Hepatitis (A through E)    -   Hospital acquired infections—for example Clostridium difficile,        Vancomycin resistant Enterococcus, and Methicillin resistant        Staphylococcus aureus    -   Herpes simplex virus (HSV)    -   Cytomegalovirus (CMV)    -   Epstein-Ban virus (EBV)    -   Encephalitis—Japanese Encephalitis virus, Chandipura virus    -   Whooping cough—Bordetella pertussis    -   Measles—paramyxovirus    -   Meningitis—Streptococcus pneumoniae and Neisseria meningitidis    -   Anthrax—Bacillus anthracis

Some examples of genetic disorders identified with this system include:

-   -   Cystic fibrosis    -   Haemophilia    -   Sickle cell disease    -   Tay-Sachs disease    -   Haemochromatosis    -   Cerebral arteriopathy    -   Crohn's disease    -   Polycistic kidney disease    -   Congential heart disease    -   Rett syndrome

A small selection of cancers identified by the diagnostic systeminclude:

-   -   Ovarian    -   Colon carcinoma    -   Multiple endocrine neoplasia    -   Retinoblastoma    -   Turcot syndrome

The above lists are not exhaustive and the diagnostic system can beconfigured to detect a much greater variety of diseases and conditionsusing nucleic acid and proteomic analysis.

Detailed Architecture of System Components LOC Device

The LOC device 30 is central to the diagnostic system. It rapidlyperforms the four major steps of a nucleic acid based moleculardiagnostic assay, i.e. sample preparation, nucleic acid extraction,nucleic acid amplification, and detection, using a microfluidicplatform. The LOC device also has alternative uses, and these aredetailed later. As discussed above, test modules 10 and 11 can adoptmany different configurations to detect different targets Likewise, theLOC device 30 has numerous different embodiments tailored to thetarget(s) of interest. One form of the LOC device 30 is LOC device 301for fluorescent detection of target nucleic acid sequences in thepathogens of a whole blood sample. For the purposes of illustration, thestructure and operation of LOC device 301 is now described in detailwith reference to FIGS. 4 to 26 and 27 to 57.

FIG. 4 is a schematic representation of the architecture of the LOCdevice 301. For convenience, process stages shown in FIG. 4 areindicated with the reference numeral corresponding to the functionalsections of the LOC device 301 that perform that process stage. Theprocess stages associated with each of the major steps of a nucleic acidbased molecular diagnostic assay are also indicated: sample input andpreparation 288, extraction 290, incubation 291, amplification 292 anddetection 294. The various reservoirs, chambers, valves and othercomponents of the LOC device 301 will be described in more detail later.

FIG. 5 is a perspective view of the LOC device 301. It is fabricatedusing high volume CMOS and MST (microsystems technology) manufacturingtechniques. The laminar structure of the LOC device 301 is illustratedin the schematic (not to scale) partial section view of FIG. 12. The LOCdevice 301 has a silicon substrate 84 which supports the CMOS+MST chip48, comprising CMOS circuitry 86 and an MST layer 87, with a cap 46overlaying the MST layer 87. For the purposes of this patentspecification, the term ‘MST layer’ is a reference to a collection ofstructures and layers that process the sample with various reagents.Accordingly, these structures and components are configured to defineflow-paths with characteristic dimensions that will support capillarydriven flow of liquids with physical characteristics similar to those ofthe sample during processing. In light of this, the MST layer andcomponents are typically fabricated using surface micromachiningtechniques and/or bulk micromachining techniques. However, otherfabrication methods can also produce structures and componentsdimensioned for capillary driven flows and processing very smallvolumes. The specific embodiments described in this specification showthe MST layer as the structures and active components supported on theCMOS circuitry 86, but excluding the features of the cap 46. However,the skilled addressee will appreciate that the MST layer need not haveunderlying CMOS or indeed an overlying cap in order for it to processthe sample.

The overall dimensions of the LOC device shown in the following figuresare 1760 μm×5824 μm. Of course, LOC devices fabricated for differentapplications may have different dimensions.

FIG. 6 shows the features of the MST layer 87 superimposed with thefeatures of the cap. Insets AA to AD, AG and AH shown in FIG. 6 areenlarged in FIGS. 13, 14, 35, 56, 55 and 58, respectively, and describedin detail below for a comprehensive understanding of each structurewithin the LOC device 301. FIGS. 7 to 10 show the features of the cap 46in isolation while FIG. 11 shows the CMOS+MST device 48 structures inisolation.

Laminar Structure

FIGS. 12 and 22 are sketches that diagrammatically show the laminarstructure of the CMOS+MST device 48, the cap 46 and the fluidicinteraction between the two. The figures are not to scale for thepurposes of illustration. FIG. 12 is a schematic section view throughthe sample inlet 68 and FIG. 22 is a schematic section through thereservoir 54. As best shown in FIG. 12, the CMOS+MST device 48 has asilicon substrate 84 which supports the CMOS circuitry 86 that operatesthe active elements within the MST layer 87 above. A passivation layer88 seals and protects the CMOS layer 86 from the fluid flows through theMST layer 87.

Fluid flows through both the cap channels 94 and the MST channels 90(see for example FIGS. 7 and 16) in the cap layer 46 and MST channellayer 100, respectively. Cell transport occurs in the larger channels 94fabricated in the cap 46, while biochemical processes are carried out inthe smaller MST channels 90. Cell transport channels are sized so as tobe able to transport cells in the sample to predetermined sites in theMST channels 90. Transportation of cells with sizes greater than 20microns (for example, certain leukocytes) requires channel dimensionsgreater than 20 microns, and therefore a cross sectional area transverseto the flow of greater than 400 square microns. MST channels,particularly at locations in the LOC where transport of cells is notrequired, can be significantly smaller.

It will be appreciated that cap channel 94 and MST channel 90 aregeneric references and particular MST channels 90 may also be referredto as (for example) heated microchannels or dialysis MST channels inlight of their particular function. MST channels 90 are formed byetching through a MST channel layer 100 deposited on the passivationlayer 88 and patterned with photoresist. The MST channels 90 areenclosed by a roof layer 66 which forms the top (with respect to theorientation shown in the figures) of the CMOS+MST device 48.

Despite sometimes being shown as separate layers, the cap channel layer80 and the reservoir layer 78 are formed from a unitary piece ofmaterial. Of course, the piece of material may also be non-unitary. Thispiece of material is etched from both sides in order to form a capchannel layer 80 in which the cap channels 94 are etched and thereservoir layer 78 in which the reservoirs 54, 56, 58, 60 and 62 areetched. Alternatively, the reservoirs and the cap channels are formed bya micromolding process. Both etching and micromolding techniques areused to produce channels with cross sectional areas transverse to theflow as large as 20,000 square microns, and as small as 8 squaremicrons.

At different locations in the LOC device, there can be a range ofappropriate choices for the cross sectional area of the channeltransverse to the flow. Where large quantities of sample, or sampleswith large constituents, are contained in the channel, a cross-sectionalarea of up to 20,000 square microns (for example, a 200 micron widechannel in a 100 micron thick layer) is suitable. Where small quantitiesof liquid, or mixtures without large cells present, are contained in thechannel, a very small cross sectional area transverse to the flow ispreferable.

A lower seal 64 encloses the cap channels 94 and the upper seal layer 82encloses the reservoirs 54, 56, 58, 60 and 62.

The five reservoirs 54, 56, 58, 60 and 62 are preloaded withassay-specific reagents. In the embodiment described here, thereservoirs are preloaded with the following reagents, but other reagentscan easily be substituted:

-   -   reservoir 54: anticoagulant with option to include erythrocyte        lysis buffer    -   reservoir 56: lysis reagent    -   reservoir 58: restriction enzymes, ligase and linkers (for        linker-primed PCR (see FIG. 61, extracted from T. Stachan et        al., Human Molecular Genetics 2, Garland Science, NY and London,        1999))    -   reservoir 60: amplification mix (dNTPs, primers, buffer) and    -   reservoir 62: DNA polymerase.

The cap 46 and the CMOS+MST layers 48 are in fluid communication viacorresponding openings in the lower seal 64 and the roof layer 66. Theseopenings are referred to as uptakes 96 and downtakes 92 depending onwhether fluid is flowing from the MST channels 90 to the cap channels 94or vice versa.

LOC Device Operation

The operation of the LOC device 301 is described below in a step-wisefashion with reference to analysing pathogenic DNA in a blood sample. Ofcourse, other types of biological or non-biological fluid are alsoanalysed using an appropriate set, or combination, of reagents, testprotocols, LOC variants and detection systems. Referring back to FIG. 4,there are five major steps involved in analysing a biological sample,comprising sample input and preparation 288, nucleic acid extraction290, nucleic acid incubation 291, nucleic acid amplification 292 anddetection and analysis 294.

The sample input and preparation step 288 involves mixing the blood withan anticoagulant 116 and then separating pathogens from the leukocytesand erythrocytes with the pathogen dialysis section 70. As best shown inFIGS. 7 and 12, the blood sample enters the device via the sample inlet68. Capillary action draws the blood sample along the cap channel 94 tothe reservoir 54. Anticoagulant is released from the reservoir 54 as thesample blood flow opens its surface tension valve 118 (see FIGS. 15 and22). The anticoagulant prevents the formation of clots which would blockthe flow.

As best shown in FIG. 22, the anticoagulant 116 is drawn out of thereservoir 54 by capillary action and into the MST channel 90 via thedowntake 92. The downtake 92 has a capillary initiation feature (CIF)102 to shape the geometry of the meniscus such that it does not anchorto the rim of the downtake 92. Vent holes 122 in the upper seal 82allows air to replace the anticoagulant 116 as it is drawn out of thereservoir 54.

The MST channel 90 shown in FIG. 22 is part of a surface tension valve118. The anticoagulant 116 fills the surface tension valve 118 and pinsa meniscus 120 to the uptake 96 to a meniscus anchor 98. Prior to use,the meniscus 120 remains pinned at the uptake 96 so the anticoagulantdoes not flow into the cap channel 94. When the blood flows through thecap channel 94 to the uptake 96, the meniscus 120 is removed and theanticoagulant is drawn into the flow.

FIGS. 15 to 21 show Inset AE which is a portion of Inset AA shown inFIG. 13. As shown in FIGS. 15, 16 and 17, the surface tension valve 118has three separate MST channels 90 extending between respectivedowntakes 92 and uptakes 96. The number of MST channels 90 in a surfacetension valve can be varied to change the flow rate of the reagent intothe sample mixture. As the sample mixture and the reagents mix togetherby diffusion, the flow rate out of the reservoir determines theconcentration of the reagent in the sample flow. Hence, the surfacetension valve for each of the reservoirs is configured to match thedesired reagent concentration.

The blood passes into a pathogen dialysis section 70 (see FIGS. 4 and15) where target cells are concentrated from the sample using an arrayof apertures 164 sized according to a predetermined threshold. Cellssmaller than the threshold pass through the apertures while larger cellsdo not pass through the apertures. Unwanted cells, which may be eitherthe larger cells withheld by the array of apertures 164 or the smallercells that pass through the apertures, are redirected to a waste unit 76while the target cells continue as part of the assay.

In the pathogen dialysis section 70 described here, the pathogens fromthe whole blood sample are concentrated for microbial DNA analysis. Thearray of apertures is formed by a multitude of 3 micron diameter holes164 fluidically connecting the input flow in the cap channel 94 to atarget channel 74. The 3 micron diameter apertures 164 and the dialysisuptake holes 168 for the target channel 74 are connected by a series ofdialysis MST channels 204 (best shown in FIGS. 15 and 21). Pathogens aresmall enough to pass through the 3 micron diameter apertures 164 andfill the target channel 74 via the dialysis MST channels 204. Cellslarger than 3 microns, such as erythrocytes and leukocytes, stay in thewaste channel 72 in the cap 46 which leads to a waste reservoir 76 (seeFIG. 7).

Other aperture shapes, sizes and aspect ratios can be used to isolatespecific pathogens or other target cells such as leukocytes for humanDNA analysis. Greater detail on the dialysis section and dialysisvariants is provided later.

Referring again to FIGS. 6 and 7, the flow is drawn through the targetchannel 74 to the surface tension valve 128 of the lysis reagentreservoir 56. The surface tension valve 128 has seven MST channels 90extending between the lysis reagent reservoir 56 and the target channel74. When the menisci are unpinned by the sample flow, the flow rate fromall seven of the MST channels 90 will be greater than the flow rate fromthe anticoagulant reservoir 54 where the surface tension valve 118 hasthree MST channels 90 (assuming the physical characteristics of thefluids are roughly equivalent). Hence the proportion of lysis reagent inthe sample mixture is greater than that of the anticoagulant.

The lysis reagent and target cells mix by diffusion in the targetchannel 74 within the chemical lysis section 130. A boiling-initiatedvalve 126 stops the flow until sufficient time has passed for diffusionand lysis to take place, releasing the genetic material from the targetcells (see FIGS. 6 and 7). The structure and operation of theboiling-initiated valves are described in greater detail below withreference to FIGS. 31 and 32. Other active valve types (as opposed topassive valves such as the surface tension valve 118) have also beendeveloped by the Applicant which may be used here instead of theboiling-initiated valve. These alternative valve designs are alsodescribed later.

When the boiling-initiated valve 126 opens, the lysed cells flow into amixing section 131 for pre-amplification restriction digestion andlinker ligation.

Referring to FIG. 13, restriction enzymes, linkers and ligase arereleased from the reservoir 58 when the flow unpins the menisci at thesurface tension valve 132 at the start of the mixing section 131. Themixture flows the length of the mixing section 131 for diffusion mixing.At the end of the mixing section 131 is downtake 134 leading into theincubator inlet channel 133 of the incubation section 114 (see FIG. 13).The incubator inlet channel 133 feeds the mixture into a serpentineconfiguration of heated microchannels 210 which provides an incubationchamber for holding the sample during restriction digestion and ligationof the linkers (see FIGS. 13 and 14).

FIGS. 23, 24, 25, 26, 27, 28 and 29 show the layers of the LOC device301 within Inset AB of FIG. 6. Each figure shows the sequential additionof layers forming the structures of the CMOS+MST layer 48 and the cap46. Inset AB shows the end of the incubation section 114 and the startof the amplification section 112. As best shown in FIGS. 14 and 23, theflow fills the microchannels 210 of the incubation section 114 untilreaching the boiling-initiated valve 106 where the flow stops whilediffusion takes place. As discussed above, the microchannel 210 upstreamof the boiling-initiated valve 106 becomes an incubation chambercontaining the sample, restriction enzymes, ligase and linkers. Theheaters 154 are then activated and held at constant temperature for aspecified time for restriction digestion and linker ligation to occur.

The skilled worker will appreciate that this incubation step 291 (seeFIG. 4) is optional and only required for some nucleic acidamplification assay types. Furthermore, in some instances, it may benecessary to have a heating step at the end of the incubation period tospike the temperature above the incubation temperature. The temperaturespike inactivates the restriction enzymes and ligase prior to enteringthe amplification section 112. Inactivation of the restriction enzymesand ligase has particular relevance when isothermal nucleic acidamplification is being employed.

Following incubation, the boiling-initiated valve 106 is activated(opened) and the flow resumes into the amplification section 112.Referring to FIGS. 31 and 32, the mixture fills the serpentineconfiguration of heated microchannels 158, which form one or moreamplification chambers, until it reaches the boiling-initiated valve108. As best shown in the schematic section view of FIG. 30,amplification mix (dNTPs, primers, buffer) is released from reservoir 60and polymerase is subsequently released from reservoir 62 into theintermediate MST channel 212 connecting the incubation and amplificationsections (114 and 112 respectively).

FIGS. 35 to 51 show the layers of the LOC device 301 within Inset AC ofFIG. 6. Each figure shows the sequential addition of layers forming thestructures of the CMOS+MST device 48 and the cap 46. Inset AC is at theend of the amplification section 112 and the start of the hybridizationand detection section 52. The incubated sample, amplification mix andpolymerase flow through the microchannels 158 to the boiling-initiatedvalve 108. After sufficient time for diffusion mixing, the heaters 154in the microchannels 158 are activated for thermal cycling or isothermalamplification. The amplification mix goes through a predetermined numberof thermal cycles or a preset amplification time to amplify sufficienttarget DNA. After the nucleic acid amplification process, theboiling-initiated valve 108 opens and flow resumes into thehybridization and detection section 52. The operation ofboiling-initiated valves is described in more detail later.

As shown in FIG. 52, the hybridization and detection section 52 has anarray of hybridization chambers 110. FIGS. 52, 53, 54 and 56 show thehybridization chamber array 110 and individual hybridization chambers180 in detail. At the entrance to the hybridization chamber 180 is adiffusion barrier 175 which prevents diffusion of the target nucleicacid, probe strands and hybridized probes between the hybridizationchambers 180 during hybridization so as to prevent erroneoushybridization detection results. The diffusion barriers 175 present aflow-path-length that is long enough to prevent the target sequences andprobes diffusing out of one chamber and contaminating another chamberwithin the time taken for the probes and nucleic acids to hybridize andthe signal to be detected, thus avoiding an erroneous result.

Another mechanism to prevent erroneous readings is to have identicalprobes in a number of the hybridization chambers. The CMOS circuitry 86derives a single result from the photodiodes 184 corresponding to thehybridization chambers 180 that contain identical probes. Anomalousresults can be disregarded or weighted differently in the derivation ofthe single result.

The thermal energy required for hybridization is provided byCMOS-controlled heaters 182 (described in more detail below). After theheater is activated, hybridization occurs between complementarytarget-probe sequences. The LED driver 29 in the CMOS circuitry 86signals the LED 26 located in the test module 10 to illuminate. Theseprobes only fluoresce when hybridization has occurred thereby avoidingwashing and drying steps that are typically required to remove unboundstrands. Hybridization forces the stem-and-loop structure of the FRETprobes 186 to open, which allows the fluorophore to emit fluorescentenergy in response to the LED excitation light, as discussed in greaterdetail later. Fluorescence is detected by a photodiode 184 in the CMOScircuitry 86 underlying each hybridization chamber 180 (seehybridization chamber description below). The photodiodes 184 for allhybridization chambers and associated electronics collectively form thephotosensor 44 (see FIG. 59). In other embodiments, the photosensor maybe an array of charge coupled devices (CCD array). The detected signalfrom the photodiodes 184 is amplified and converted to a digital outputwhich is analyzed by the test module reader 12. Further details of thedetection method are described later.

Additional Details for the LOC Device Modularity of the Design

The LOC device 301 has many functional sections, including the reagentreservoirs 54, 56, 58, 60 and 62, the dialysis section 70, lysis section130, incubation section 114, and amplification section 112, valve types,the humidifier and humidity sensor. In other embodiments of the LOCdevice, these functional sections can be omitted, additional functionalsections can be added or the functional sections can be used foralternative purposes to those described above.

For example, the incubation section 114 can be used as the firstamplification section 112 of a tandem amplification assay system, withthe chemical lysis reagent reservoir 56 being used to add the firstamplification mix of primers, dNTPs and buffer and reagent reservoir 58being used for adding the reverse transcriptase and/or polymerase. Achemical lysis reagent can also be added to the reservoir 56 along withthe amplification mix if chemical lysis of the sample is desired or,alternatively, thermal lysis can occur in the incubation section byheating the sample for a predetermined time. In some embodiments, anadditional reservoir can be incorporated immediately upstream ofreservoir 58 for the mix of primers, dNTPs and buffer if there is arequirement for chemical lysis and a separation of this mix from thechemical lysis reagent is desired.

In some circumstances it may be desirable to omit a step, such as theincubation step 291. In this case, a LOC device can be specificallyfabricated to omit the reagent reservoir 58 and incubation section 114,or the reservoir can simply not be loaded with reagents or the activevalves, if present, not activated to dispense the reagents into thesample flow, and the incubation section then simply becomes a channel totransport the sample from the lysis section 130 to the amplificationsection 112. The heaters are independently operable and therefore, wherereactions are dependent on heat, such as thermal lysis, programming theheaters not to activate during this step ensures thermal lysis does notoccur in LOC devices that do not require it. The dialysis section 70 canbe located at the beginning of the fluidic system within themicrofluidic device as shown in FIG. 4 or can be located anywhere elsewithin the microfluidic device. For example, dialysis after theamplification phase 292 to remove cellular debris prior to thehybridization and detection step 294 may be beneficial in somecircumstances. Alternatively, two or more dialysis sections can beincorporated at any location throughout the LOC device. Similarly, it ispossible to incorporate additional amplification sections 112 to enablemultiple targets to be amplified in parallel or in series prior to beingdetected in the hybridization chamber arrays 110 with specific nucleicacid probes. For analysis of samples like whole blood, in which dialysisis not required, the dialysis section 70 is simply omitted from thesample input and preparation section 288 of the LOC design. In somecases, it is not necessary to omit the dialysis section 70 from the LOCdevice even if the analysis does not require dialysis. If there is nogeometric hindrance to the assay by the existence of a dialysis section,a LOC with the dialysis section 70 in the sample input and preparationsection can still be used without a loss of the required functionality.

Furthermore, the detection section 294 may encompass proteomic chamberarrays which are identical to the hybridization chamber arrays but areloaded with probes designed to conjugate or hybridize with sample targetproteins present in non-amplified sample instead of nucleic acid probesdesigned to hybridize to target nucleic acid sequences.

It will be appreciated that the LOC devices fabricated for use in thisdiagnostic system are different combinations of functional sectionsselected in accordance with the particular LOC application. The vastmajority of functional sections are common to many of the LOC devicesand the design of additional LOC devices for new application is a matterof compiling an appropriate combination of functional sections from theextensive selection of functional sections used in the existing LOCdevices.

Only a small number of the LOC devices are shown in this description andsome more are shown schematically to illustrate the design flexibilityof the LOC devices fabricated for this system. The person skilled in theart will readily recognise that the LOC devices shown in thisdescription are not an exhaustive list and many additional LOC designsare a matter of compiling the appropriate combination of functionalsections.

Sample Types

LOC variants can accept and analyze the nucleic acid or protein contentof a variety of sample types in liquid form including, but not limitedto, blood and blood products, saliva, cerebrospinal fluid, urine, semen,amniotic fluid, umbilical cord blood, breast milk, sweat, pleuraleffusion, tear, pericardial fluid, peritoneal fluid, environmental watersamples and drink samples. Amplicon obtained from macroscopic nucleicacid amplification can also be analysed using the LOC device; in thiscase, all the reagent reservoirs will be empty or configured not torelease their contents, and the dialysis, lysis, incubation andamplification sections will be used solely to transport the sample fromthe sample inlet 68 to the hybridization chambers 180 for nucleic aciddetection, as described above.

For some sample types, a pre-processing step is required, for examplesemen may need to be liquefied and mucus may need to be pre-treated withan enzyme to reduce the viscosity prior to input into the LOC device.

Sample Unit

Referring to FIGS. 1 and 12, the sample is added to the macroreceptacle24 of the test module 10. The macroreceptacle 24 is a truncated conewhich feeds into the inlet 68 of the LOC device 301 by capillary action.Here it flows into the 64 μm wide×60 μm deep cap channel 94 where it isdrawn towards the anticoagulant reservoir 54, also by capillary action.

Reagent Reservoirs

The small volumes of reagents required by the assay systems usingmicrofluidic devices, such as LOC device 301, permit the reagentreservoirs to contain all reagents necessary for the biochemicalprocessing with each of the reagent reservoirs having a small volume.This volume is easily less than 1,000,000,000 cubic microns, in the vastmajority of cases less than 300,000,000 cubic microns, typically lessthan 70,000,000 cubic microns and in the case of the LOC device 301shown in the drawings, less than 20,000,000 cubic microns.

Dialysis Section

Referring to FIGS. 15 to 21, 33 and 34, the pathogen dialysis section 70is designed to concentrate pathogenic target cells from the sample. Aspreviously described, a plurality of apertures in the form of 3 microndiameter holes 164 in the roof layer 66 filter the target cells from thebulk of the sample. As the sample flows past the 3 micron diameterapertures 164, microbial pathogens pass through the holes into a seriesof dialysis MST channels 204 and flow back up into the target channel 74via 16 μm dialysis uptake holes 168 (see FIGS. 33 and 34). The remainderof the sample (erythrocytes and so on) stay in the cap channel 94.Downstream of the pathogen dialysis section 70, the cap channel 94becomes the waste channel 72 leading to the waste reservoir 76. Forbiological samples of the type that generate a substantial amount ofwaste, a foam insert or other porous element 49 within the outer casing13 of the test module 10 is configured to be in fluid communication withthe waste reservoir 76 (see FIG. 1).

The pathogen dialysis section 70 functions entirely on capillary actionof the fluid sample. The 3 micron diameter apertures 164 at the upstreamend of the pathogen dialysis section 70 have capillary initiationfeatures (CIFs) 166 (see FIG. 33) so that the fluid is drawn down intothe dialysis MST channel 204 beneath. The first uptake hole 198 for thetarget channel 74 also has a CIF 202 (see FIG. 15) to avoid the flowsimply pinning a meniscus across the dialysis uptake holes 168.

The small constituents dialysis section 682 schematically shown in FIG.70 can have a similar structure to the pathogen dialysis section 70. Thesmall constituents dialysis section separates any small target cells ormolecules from a sample by sizing (and, if necessary, shaping) aperturessuitable for allowing the small target cells or molecules to pass intothe target channel and continue for further analysis. Larger sized cellsor molecules are removed to a waste reservoir 766. Thus, the LOC device30 (see FIGS. 1 and 85) is not limited to separating pathogens that areless than 3 μm in size, but can be used to separate cells or moleculesof any size desired.

Lysis Section

Referring back to FIGS. 7, 11 and 13, the genetic material in the sampleis released from the cells by a chemical lysis process. As describedabove, a lysis reagent from the lysis reservoir 56 mixes with the sampleflow in the target channel 74 downstream of the surface tension valve128 for the lysis reservoir 56. However, some diagnostic assays arebetter suited to a thermal lysis process, or even a combination ofchemical and thermal lysis of the target cells. The LOC device 301accommodates this with the heated microchannels 210 of the incubationsection 114. The sample flow fills the incubation section 114 and stopsat the boiling-initiated valve 106. The incubation microchannels 210heat the sample to a temperature at which the cellular membranes aredisrupted.

In some thermal lysis applications, an enzymatic reaction in thechemical lysis section 130 is not necessary and the thermal lysiscompletely replaces the enzymatic reaction in the chemical lysis section130.

Boiling-Initiated Value

As discussed above, the LOC device 301 has three boiling-initiatedvalves 126, 106 and 108. The location of these valves is shown in FIG.6. FIG. 31 is an enlarged plan view of the boiling-initiated valve 108in isolation at the end of the heated microchannels 158 of theamplification section 112.

The sample flow 119 is drawn along the heated microchannels 158 bycapillary action until it reaches the boiling-initiated valve 108. Theleading meniscus 120 of the sample flow pins at a meniscus anchor 98 atthe valve inlet 146. The geometry of the meniscus anchor 98 stops theadvancing meniscus to arrest the capillary flow. As shown in FIGS. 31and 32, the meniscus anchor 98 is an aperture provided by an uptakeopening from the MST channel 90 to the cap channel 94. Surface tensionin the meniscus 120 keeps the valve closed. An annular heater 152 is atthe periphery of the valve inlet 146. The annular heater 152 isCMOS-controlled via the boiling-initiated valve heater contacts 153.

To open the valve, the CMOS circuitry 86 sends an electrical pulse tothe valve heater contacts 153. The annular heater 152 resistively heatsuntil the liquid sample 119 boils. The boiling unpins the meniscus 120from the valve inlet 146 and initiates wetting of the cap channel 94.Once wetting the cap channel 94 begins, capillary flow resumes. Thefluid sample 119 fills the cap channel 94 and flows through the valvedowntake 150 to the valve outlet 148 where capillary driven flowcontinues along the amplification section exit channel 160 into thehybridization and detection section 52. Liquid sensors 174 are placedbefore and after the valve for diagnostics.

It will be appreciated that once the boiling-initiated valves areopened, they cannot be re-closed. However, as the LOC device 301 and thetest module 10 are single-use devices, re-closing the valves isunnecessary.

Incubation Section and Nucleic Acid Amplification Section

FIGS. 6, 7, 13, 14, 23, 24, 25, 35 to 45, 50 and 51 show the incubationsection 114 and the amplification section 112. The incubation section114 has a single, heated incubation microchannel 210 etched in aserpentine pattern in the MST channel layer 100 from the downtakeopening 134 to the boiling-initiated valve 106 (see FIGS. 13 and 14).Control over the temperature of the incubation section 114 enablesenzymatic reactions to take place with greater efficiency. Similarly,the amplification section 112 has a heated amplification microchannel158 in a serpentine configuration leading from the boiling-initiatedvalve 106 to the boiling-initiated valve 108 (see FIGS. 6 and 14). Thesevalves arrest the flow to retain the target cells in the heatedincubation or amplification microchannels 210 or 158 while mixing,incubation and nucleic acid amplification takes place. The serpentinepattern of the microchannels also facilitates (to some extent) mixing ofthe target cells with reagents.

In the incubation section 114 and the amplification section 112, thesample cells and the reagents are heated by the heaters 154 controlledby the CMOS circuitry 86 using pulse width modulation (PWM). Eachmeander of the serpentine configuration of the heated incubationmicrochannel 210 and amplification microchannel 158 has three separatelyoperable heaters 154 extending between their respective heater contacts156 (see FIG. 14) which provides for the two-dimensional control ofinput heat flux density. As best shown in FIG. 51, the heaters 154 aresupported on the roof layer 66 and embedded in the lower seal 64. Theheater material is TiAl but many other conductive metals would besuitable. The elongate heaters 154 are parallel with the longitudinalextent of each channel section that forms the wide meanders of theserpentine shape. In the amplification section 112, each of the widemeanders can operate as separate PCR chambers via individual heatercontrol.

The small volumes of amplicon required by the assay systems usingmicrofluidic devices, such as LOC device 301, permit low amplificationmixture volumes for amplification in amplification section 112. Thisvolume is easily less than 400 nanoliters, in the vast majority of casesless than 170 nanoliters, typically less than 70 nanoliters and in thecase of the LOC device 301, between 2 nanoliters and 30 nanoliters.

Increased Rates of Heating and Greater Diffusive Mixing

The small cross section of each channel section increases the heatingrate of the amplification fluid mix. All the fluid is kept a relativelyshort distance from the heater 154. Reducing the channel cross section(that is the amplification microchannel 158 cross section) to less than100,000 square microns achieves appreciably higher heating rates thanthat provided by more ‘macro-scale’ equipment. Lithographic fabricationtechniques allow the amplification microchannel 158 to have a crosssectional area transverse to the flow-path less than 16,000 squaremicrons which gives substantially higher heating rates. Feature sizes onthe order of 1 micron are readily achievable with lithographictechniques. If very little amplicon is needed (as is the case in the LOCdevice 301), the cross sectional area can be reduced to less than 2,500square microns. For diagnostic assays with 1,000 to 2,000 probes on theLOC device, and a requirement of ‘sample-in, answer out’ in less than 1minute, a cross sectional area transverse to the flow of between 400square microns and 1 square micron is adequate.

The heater element in the amplification microchannel 158 heats thenucleic acid sequences at a rate more than 80 Kelvin (K) per second, inthe vast majority of cases at a rate greater than 100 K per second.Typically, the heater element heats the nucleic acid sequences at a ratemore than 1,000 K per second and in many cases, the heater element heatsthe nucleic acid sequences at a rate more than 10,000 K per second.Commonly, based on the demands of the assay system, the heater elementheats the nucleic acid sequences at a rate more than 100,000 K persecond, more than 1,000,000 K per second more than 10,000,000 K persecond, more than 20,000,000 K per second, more than 40,000,000 K persecond, more than 80,000,000 K per second and more than 160,000,000 Kper second.

A small cross-sectional area channel is also beneficial for diffusivemixing of any reagents with the sample fluid. Before diffusive mixing iscomplete, diffusion of one liquid into the other is greatest near theinterface between the two. Concentration decreases with distance fromthe interface. Using microchannels with relatively small cross sectionstransverse to the flow direction, keeps both fluid flows close to theinterface for more rapid diffusive mixing. Reducing the channel crosssection to less than 100,000 square microns achieves appreciably highermixing rates than that provided by more ‘macro-scale’ equipment.Lithographic fabrication techniques allows microchannels with a crosssectional area transverse to the flow-path less than 16000 squaremicrons which gives significantly higher mixing rates. If small volumesare needed (as is the case in the LOC device 301), the cross sectionalarea can be reduced to less than 2500 square microns. For diagnosticassays with 1000 to 2000 probes on the LOC device, and a requirement of‘sample-in, answer out’ in less than 1 minute, a cross sectional areatransverse to the flow of between 400 square microns and 1 square micronis adequate.

Short Thermal Cycle Times

Keeping the sample mixture proximate to the heaters, and using verysmall fluid volumes allows rapid thermal cycling during the nucleic acidamplification process. Each thermal cycle (i.e. denaturing, annealingand primer extension) is completed in less than 30 seconds for targetsequences up to 150 base pairs (bp) long. In the vast majority ofdiagnostic assays, the individual thermal cycle times are less than 11seconds, and a large proportion are less than 4 seconds. LOC devices 30with some of the most common diagnostic assays have thermal cycles timebetween 0.45 seconds to 1.5 seconds for target sequences up to 150 bplong. Thermal cycling at this rate allows the test module to completethe nucleic acid amplification process in much less than 10 minutes;often less than 220 seconds. For most assays, the amplification sectiongenerates sufficient amplicon in less than 80 seconds from the samplefluid entering the sample inlet. For a great many assays, sufficientamplicon is generated in 30 seconds.

Upon completion of a preset number of amplification cycles, the ampliconis fed into the hybridization and detection section 52 via theboiling-initiated valve 108.

Hybridization Chambers

FIGS. 52, 53, 54, 56 and 57 show the hybridization chambers 180 in thehybridization chamber array 110. The hybridization and detection section52 has a 24×45 array 110 of hybridization chambers 180, each withhybridization-responsive FRET probes 186, heater element 182 and anintegrated photodiode 184. The photodiode 184 is incorporated fordetection of fluorescence resulting from the hybridization of a targetnucleic acid sequence or protein with the FRET probes 186. Eachphotodiode 184 is independently controlled by the CMOS circuitry 86. Anymaterial between the FRET probes 186 and the photodiode 184 must betransparent to the emitted light. Accordingly, the wall section 97between the probes 186 and the photodiode 184 is also opticallytransparent to the emitted light. In the LOC device 301, the wallsection 97 is a thin (approximately 0.5 micron) layer of silicondioxide.

Incorporation of a photodiode 184 directly beneath each hybridizationchamber 180 allows the volume of probe-target hybrids to be very smallwhile still generating a detectable fluorescence signal (see FIG. 54).The small amounts permit small volume hybridization chambers. Adetectable amount of probe-target hybrid requires a quantity of probe,prior to hybridization, which is easily less than 270 picograms(corresponding to 900,000 cubic microns), in the vast majority of casesless than 60 picograms (corresponding to 200,000 cubic microns),typically less than 12 picograms (corresponding to 40,000 cubic microns)and in the case of the LOC device 301 shown in the accompanying figures,less than 2.7 picograms (corresponding to a chamber volume of 9,000cubic microns). Of course, reducing the size of the hybridizationchambers allows a higher density of chambers and therefore more probeson the LOC device. In LOC device 301, the hybridization section has morethan 1,000 chambers in an area of 1,500 microns by 1,500 microns (i.e.less than 2,250 square microns per chamber). Smaller volumes also reducethe reaction times so that hybridization and detection is faster. Anadditional advantage of the small amount of probe required in eachchamber is that only very small quantities of probe solution need to bespotted into each chamber during production of the LOC device.Embodiments of the LOC device according to the invention can be spottedusing a probe solution volume of 1 picoliter or less.

After nucleic acid amplification, boiling-initiated valve 108 isactivated and the amplicon flows along the flow-path 176 and into eachof the hybridization chambers 180 (see FIGS. 52 and 56). An end-pointliquid sensor 178 indicates when the hybridization chambers 180 arefilled with amplicon and the heaters 182 can be activated.

After sufficient hybridization time, the LED 26 (see FIG. 2) isactivated. The opening in each of the hybridization chambers 180provides an optical window 136 for exposing the FRET probes 186 to theexcitation radiation (see FIGS. 52, 54 and 56). The LED 26 isilluminated for a sufficiently long time in order to induce afluorescence signal from the probes with high intensity. Duringexcitation, the photodiode 184 is shorted. After a pre-programmed delay300 (see FIG. 2), the photodiode 184 is enabled and fluorescenceemission is detected in the absence of the excitation light. Theincident light on the active area 185 of the photodiode 184 (see FIG.54) is converted into a photocurrent which can then be measured usingCMOS circuitry 86.

The hybridization chambers 180 are each loaded with probes for detectinga single target nucleic acid sequence. Each hybridization chambers 180can be loaded with probes to detect over 1,000 different targets ifdesired. Alternatively, many or all the hybridization chambers can beloaded with the same probes to detect the same target nucleic acidrepeatedly. Replicating the probes in this way throughout thehybridization chamber array 110 leads to increased confidence in theresults obtained and the results can be combined by the photodiodesadjacent those hybridization chambers to provide a single result ifdesired. The person skilled in the art will recognise that it ispossible to have from one to over 1,000 different probes on thehybridization chamber array 110, depending on the assay specification.

Hybridization Chambers with Electrochemiluminescence Detection

FIGS. 78, 90, 115 and 116 show the hybridization chambers 180 used in anECL variant of the LOC device, LOC variant L 729. In this embodiment ofthe LOC device, a 24×45 array 110 of hybridization chambers 180, eachwith hybridization-responsive ECL probes 237, is positioned inregistration with a corresponding array of photodiodes 184 integratedinto the CMOS. In a similar fashion to the LOC devices configured forfluorescence detection, each photodiode 184 is incorporated fordetection of ECL resulting from the hybridization of a target nucleicacid sequence or protein with an ECL probe 237. Each photodiode 184 isindependently controlled by the CMOS circuitry 86. Again, thetransparent wall section 97 between the probes 186 and the photodiode184 is transparent to the emitted light.

A photodiode 184 closely adjacent each hybridization chamber 180 allowsthe amount of probe-target hybrids to be very small while stillgenerating a detectable ECL signal (see FIG. 78). The small amountspermit small volume hybridization chambers. A detectable amount ofprobe-target hybrid requires a quantity of probe, prior tohybridization, which is easily less than 270 picograms (corresponding toa chamber volume of 900,000 cubic microns), in the vast majority ofcases less than 60 picograms (corresponding to 200,000 cubic microns),typically less than 12 picograms (corresponding to 40,000 cubic microns)and in the case of the LOC device shown in the drawings less than 2.7picograms (corresponding to a chamber volume of 9,000 cubic microns). Ofcourse, reducing the size of the hybridization chambers allows a higherdensity of chambers and therefore more probes on the LOC device. In theLOC device shown, the hybridization section has more than 1,000 chambersin an area of 1,500 microns by 1,500 microns (i.e. less than 2,250square microns per chamber). Smaller volumes also reduce the reactiontimes so that hybridization and detection is faster. An additionaladvantage of the small amount of probe required in each chamber is thatonly very small quantities of probe solution need be spotted into eachchamber during production of the LOC device. In the case of the LOCdevice shown in the drawings, the required amount of probe can bespotted using a solution volume of 1 picoliter or less.

After nucleic acid amplification, the boiling-initiated valve 108 isactivated and the amplicon flows along the flow-path 176 and into eachof the hybridization chambers 180 (see FIGS. 52 and 116). An end-pointliquid sensor 178 indicates when the hybridization chambers 180 arefilled with amplicon so that the heaters 182 can be activated.

After sufficient hybridization time, the photodiode 184 is enabled readyfor collection of the ECL signal. Then the ECL excitation drivers 39(see FIG. 86) activate the ECL electrodes 860 and 870 for apredetermined length of time. The photodiode 184 remains active for ashort time after cessation of the ECL excitation current to maximize thesignal-to-noise ratio. For example, if the photodiode 184 remains activefor five times the decay lifetime of the luminescent emission, then thesignal will have decayed to less than one percent of the initial value.The incident light on the photodiode 184 is converted into aphotocurrent which can then be measured using CMOS circuitry 86.

Humidifier and Humidity Sensor

Inset AG of FIG. 6 indicates the position of the humidifier 196. Thehumidifier prevents evaporation of the reagents and probes duringoperation of the LOC device 301. As best shown in the enlarged view ofFIG. 55, a water reservoir 188 is fluidically connected to threeevaporators 190. The water reservoir 188 is filled with molecularbiology-grade water and sealed during manufacturing. As best shown inFIGS. 55 and 60, water is drawn into three downtakes 194 and alongrespective water supply channels 192 by capillary action to a set ofthree uptakes 193 at the evaporators 190. A meniscus pins at each uptake193 to retain the water. The evaporators have annular shaped heaters 191which encircle the uptakes 193. The annular heaters 191 are connected tothe CMOS circuitry 86 by the conductive columns 376 to the top metallayer 195 (see FIG. 37). Upon activation, the annular heaters 191 heatthe water causing evaporation and humidifying the device surrounds.

The position of the humidity sensor 232 is also shown in FIG. 6.However, as best shown in the enlarged view of Inset AH in FIG. 58, thehumidity sensor has a capacitive comb structure. A lithographicallyetched first electrode 296 and a lithographically etched secondelectrode 298 face each other such that their teeth are interleaved. Theopposed electrodes form a capacitor with a capacitance that can bemonitored by the CMOS circuitry 86. As the humidity increases, thepermittivity of the air gap between the electrodes increases, so thatthe capacitance also increases. The humidity sensor 232 is adjacent thehybridization chamber array 110 where humidity measurement is mostimportant to slow evaporation from the solution containing the exposedprobes.

Feedback Sensors

Temperature and liquid sensors are incorporated throughout the LOCdevice 301 to provide feedback and diagnostics during device operation.Referring to FIG. 35, nine temperature sensors 170 are distributedthroughout the amplification section 112 Likewise, the incubationsection 114 also has nine temperature sensors 170. These sensors eachuse a 2×2 array of bipolar junction transistors (BJTs) to monitor thefluid temperature and provide feedback to the CMOS circuitry 86. TheCMOS circuitry 86 uses this to precisely control the thermal cyclingduring the nucleic acid amplification process and any heating duringthermal lysis and incubation.

In the hybridization chambers 180, the CMOS circuitry 86 uses thehybridization heaters 182 as temperature sensors (see FIG. 56). Theelectrical resistance of the hybridization heaters 182 is temperaturedependent and the CMOS circuitry 86 uses this to derive a temperaturereading for each of the hybridization chambers 180.

The LOC device 301 also has a number of MST channel liquid sensors 174and cap channel liquid sensors 208. FIG. 35 shows a line of MST channelliquid sensors 174 at one end of every other meander in the heatedmicrochannel 158. As best shown in FIG. 37, the MST channel liquidsensors 174 are a pair of electrodes formed by exposed areas of the topmetal layer 195 in the CMOS structure 86. Liquid closes the circuitbetween the electrodes to indicate its presence at the sensor'slocation.

FIG. 25 shows an enlarged perspective of cap channel liquid sensors 208.Opposing pairs of TiA1 electrodes 218 and 220 are deposited on the rooflayer 66. Between the electrodes 218 and 220 is a gap 222 to hold thecircuit open in the absence of liquid. The presence of liquid closes thecircuit and the CMOS circuitry 86 uses this feedback to monitor theflow.

Gravitational Independence

The test modules 10 are orientation independent. They do not need to besecured to a flat stable surface in order to operate. Capillary drivenfluid flows and a lack of external plumbing into ancillary equipmentallow the modules to be truly portable and simply plugged into asimilarly portable hand held reader such as a mobile telephone. Having agravitationally independent operation means the test modules are alsoaccelerationally independent to all practical extents. They areresistant to shock and vibration and will operate on moving vehicles orwhile the mobile telephone is being carried around.

Dialysis Variants

Dialysis Section with Flow Channel to Prevent Trapped Air Bubbles

Described below is an embodiment of the LOC device referred to as LOCvariant VIII 518 and shown in FIGS. 64, 65, 66 and 67. This LOC devicehas a dialysis section that fills with the fluid sample without leavingair bubbles trapped in the channels. LOC variant VIII 518 also has anadditional layer of material referred to as an interface layer 594. Theinterface layer 594 is positioned between the cap channel layer 80 andthe MST channel layer 100 of the CMOS+MST device 48. The interface layer594 allows a more complex fluidic interconnection between the reagentreservoirs and the MST layer 87 without increasing the size of thesilicon substrate 84.

Referring to FIG. 65, the bypass channel 600 is designed to introduce atime delay in the fluid sample flow from the interface waste channel 604to the interface target channel 602. This time delay allows the fluidsample to flow through the dialysis MST channel 204 to the dialysisuptake 168 where it pins a meniscus. With a capillary initiation feature(CIF) 202 at the uptake from the bypass channel 600 to the interfacetarget channel 602, the sample fluid fills the interface target channel602 from a point upstream of all the dialysis uptakes 168 from thedialysis MST channels 204.

Without the bypass channel 600, the interface target channel 602 stillstarts filling from the upstream end, but eventually the advancingmeniscus reaches and passes over an uptake belonging to an MST channelthat has not yet filled, leading into air entrapment at that point.Trapped air reduces the sample flow rate through the leukocyte dialysissection 328.

Nucleic Acid Amplification Variants Parallel PCR

Several variants of the LOC device have multiple amplification sectionsoperating in parallel. For example, LOC variant VII 492 shown in FIG. 63has parallel amplification sections 112.1 to 112.4 which allow multiplenucleic acid amplification assays to be performed simultaneously.

LOC variant XI 746 shown in FIG. 74 also has parallel amplificationsections 112.1 to 112.4, but additionally has parallel incubationsections 114.1 to 114.4, so that the sample can be processed differentlyprior to amplification. Other LOC variants, such as LOC XIV 641 shownschematically in FIG. 69, demonstrate that the number of parallelamplification sections can be “X” number, which is only limited by thesize of the LOC device. A larger LOC device can be made to accommodategreater numbers of parallel amplification sections.

Separate amplification sections can be configured to run on differentcycle times and/or temperatures for particular target sizes orparticular amplification mix constituents. With several amplificationsections running in parallel, the LOC device can operate a multiplexnucleic acid amplification process or a uniplex amplification process ineach of the sections. In multiplex nucleic acid amplification, more thanone target sequence is amplified using more than one pair of primers. Aparallel nucleic acid amplification system with “m” chambers can run theequivalent of an n-plex amplification where n=n(1)+n(2)+ . . . +n(i)+ .. . +n(m), with n(i) being the number of different primer pairs used inthe multiplex amplification being run in chamber “i”, bearing in mindthat the SNR (signal-to-noise ratio) in the parallel amplificationsystem is higher than the n-plex amplification run in a single chambersystem. Under the special case where n(i)=1, the amplification inchamber “i” becomes just a uniplex amplification.

FDirect PCR

Traditionally, PCR requires extensive purification of the target DNAprior to preparation of the reaction mixture. However, with appropriatechanges to the chemistry and sample concentration, it is possible toperform nucleic acid amplification with minimal DNA purification, ordirect amplification. When the nucleic acid amplification process isPCR, this approach is called direct PCR. In LOC devices where nucleicacid amplification is performed at a controlled, constant temperature,the approach is direct isothermal amplification. Direct nucleic acidamplification techniques have considerable advantages for use in LOCdevices, particularly relating to simplification of the required fluidicdesign. Adjustments to the amplification chemistry for direct PCR ordirect isothermal amplification include increased buffer strength, theuse of polymerases which have high activity and processivity, andadditives which chelate with potential polymerase inhibitors. Dilutionof inhibitors present in the sample is also important.

To take advantage of direct nucleic acid amplification techniques, theLOC device designs incorporate two additional features. The firstfeature is reagent reservoirs (for example reservoir 58 in FIG. 8) whichare appropriately dimensioned to supply a sufficient quantity ofamplification reaction mix, or diluent, so that the final concentrationsof sample components which might interfere with amplification chemistryare low enough to permit successful nucleic acid amplification. Thedesired dilution of non-cellular sample components is in the range of 5×to 20×. Different LOC structures, for example the pathogen dialysissection 70 in FIG. 4, are used when appropriate to ensure that theconcentration of target nucleic acid sequences is maintained at a highenough level for amplification and detection. In this embodiment,further illustrated in FIG. 6, a dialysis section which effectivelyconcentrates pathogens small enough to be passed into the amplificationsection 292 is employed upstream of the sample extraction section 290,and rejects larger cells to a waste receptacle 76. In anotherembodiment, a dialysis section is used to selectively deplete proteinsand salts in blood plasma while retaining cells of interest.

The second LOC structural feature which supports direct nucleic acidamplification is design of channel aspect ratios to adjust the mixingratio between the sample and the amplification mix components. Forexample, to ensure dilution of inhibitors associated with the sample inthe preferred 5×-20× range through a single mixing step, the length andcross-section of the sample and reagent channels are designed such thatthe sample channel, upstream of the location where mixing is initiated,constitutes a flow impedance 4×-19× higher than the flow impedance ofthe channels through which the reagent mixture flows. Control over flowimpedances in microchannels is readily achieved through control over thedesign geometry. The flow impedance of a microchannel increases linearlywith the channel length, for a constant cross-section. Importantly formixing designs, flow impedance in microchannels depends more strongly onthe smallest cross-sectional dimension. For example, the flow impedanceof a microchannel with rectangular cross-section is inverselyproportional to the cube of the smallest perpendicular dimension, whenthe aspect ratio is far from unity.

Reverse-Transcriptase PCR (RT-PCR)

Where the sample nucleic acid species being analysed or extracted isRNA, such as from RNA viruses or messenger RNA, it is first necessary toreverse transcribe the RNA into complementary DNA (cDNA) prior to PCRamplification. The reverse transcription reaction can be performed inthe same chamber as the PCR (one-step RT-PCR) or it can be performed asa separate, initial reaction (two-step RT-PCR). In the LOC variantsdescribed herein, a one-step RT-PCR can be performed simply by addingthe reverse transcriptase to reagent reservoir 62 along with thepolymerase and programming the heaters 154 to cycle firstly for thereverse transcription step and then progress onto the nucleic acidamplification step. A two-step RT-PCR could also be easily achieved byutilizing the reagent reservoir 58 to store and dispense the buffers,primers, dNTPs and reverse transcriptase and the incubation section 114for the reverse transcription step followed by amplification in thenormal way in the amplification section 112.

Isothermal Nucleic Acid Amplification

For some applications, isothermal nucleic acid amplification is thepreferred method of nucleic acid amplification, thus avoiding the needto repetitively cycle the reaction components through varioustemperature cycles but instead maintaining the amplification section ata constant temperature, typically around 37° C. to 41° C. A number ofisothermal nucleic acid amplification methods have been described,including Strand Displacement Amplification (SDA), TranscriptionMediated Amplification (TMA), Nucleic Acid Sequence Based Amplification(NASBA), Recombinase Polymerase Amplification (RPA), Helicase-Dependentisothermal DNA Amplification (HDA), Rolling Circle Amplification (RCA),Ramification Amplification (RAM) and Loop-mediated IsothermalAmplification (LAMP), and any of these, or other isothermalamplification methods, can be employed in particular embodiments of theLOC device described herein.

In order to perform isothermal nucleic acid amplification, the reagentreservoirs 60 and 62 adjoining the amplification section will be loadedwith the appropriate reagents for the specified isothermal methodinstead of PCR amplification mix and polymerase. For example, for SDA,reagent reservoir 60 contains amplification buffer, primers and dNTPsand reagent reservoir 62 contains an appropriate nickase enzyme andExo-DNA polymerase. For RPA, reagent reservoir 60 contains theamplification buffer, primers, dNTPs and recombinase proteins, withreagent reservoir 62 containing a strand displacing DNA polymerase suchas Bsu. Similarly, for HDA, reagent reservoir 60 contains amplificationbuffer, primers and dNTPs and reagent reservoir 62 contains anappropriate DNA polymerase and a helicase enzyme to unwind the doublestranded DNA strand instead of using heat. The skilled person willappreciate that the necessary reagents can be split between the tworeagent reservoirs in any manner appropriate for the nucleic acidamplification process.

For amplification of viral nucleic acids from RNA viruses such as HIV orhepatitis C virus, NASBA or TMA is appropriate as it is unnecessary tofirst transcribe the RNA to cDNA. In this example, reagent reservoir 60is filled with amplification buffer, primers and dNTPs and reagentreservoir 62 is filled with RNA polymerase, reverse transcriptase and,optionally, RNase H.

For some forms of isothermal nucleic acid amplification it may benecessary to have an initial denaturation cycle to separate the doublestranded DNA template, prior to maintaining the temperature for theisothermal nucleic acid amplification to proceed. This is readilyachievable in all embodiments of the LOC device described herein, as thetemperature of the mix in the amplification section 112 can be carefullycontrolled by the heaters 154 in the amplification microchannels 158(see FIG. 14).

Isothermal nucleic acid amplification is more tolerant of potentialinhibitors in the sample and, as such, is generally suitable for usewhere direct nucleic acid amplification from the sample is desired.Therefore, isothermal nucleic acid amplification is sometimes useful inLOC variant XLIII 673, LOC variant XLIV 674 and LOC variant XLVII 677,amongst others, shown in FIGS. 71, 72 and 73, respectively. Directisothermal amplification may also be combined with one or morepre-amplification dialysis steps 70, 686 or 682 as shown in FIGS. 71 and73 and/or a pre-hybridization dialysis step 682 as indicated in FIG. 72to help partially concentrate the target cells in the sample beforenucleic acid amplification or remove unwanted cellular debris prior tothe sample entering the hybridization chamber array 110, respectively.The person skilled in the art will appreciate that any combination ofpre-amplification dialysis and pre-hybridization dialysis can be used.

Isothermal nucleic acid amplification can also be performed in parallelamplification sections such as those schematically represented in FIGS.63, 68 and 69, multiplexed and some methods of isothermal nucleic acidamplification, such as LAMP, are compatible with an initial reversetranscription step to amplify RNA.

Photodiode

FIG. 54 shows the photodiode 184 integrated into the CMOS circuitry 86of the LOC device 301. The photodiode 184 is fabricated as part of theCMOS circuitry 86 without additional masks or steps. This is onesignificant advantage of a CMOS photodiode over a CCD, an alternatesensing technology which could be integrated on the same chip usingnon-standard processing steps, or fabricated on an adjacent chip.On-chip detection is low cost and reduces the size of the assay system.The shorter optical path length reduces noise from the surroundingenvironment for efficient collection of the fluorescence signal andeliminates the need for a conventional optical assembly of lenses andfilters.

Quantum efficiency of the photodiode 184 is the fraction of photonsimpinging on its active area 185 that are effectively converted tophoto-electrons. For standard silicon processes, the quantum efficiencyis in the range of 0.3 to 0.5 for visible light, depending on processparameters such as the amount and absorption properties of the coverlayers.

The detection threshold of the photodiode 184 determines the smallestintensity of the fluorescence signal that can be detected. The detectionthreshold also determines the size of the photodiode 184 and hence thenumber of hybridization chambers 180 in the hybridization and detectionsection 52 (see FIG. 52). The size and number of chambers are technicalparameters that are limited by the dimensions of the LOC device (in thecase of the LOC device 301, the dimensions are 1760 μm×5824 μm) and thereal estate available after other functional modules such as thepathogen dialysis section 70 and amplification section(s) 112 areincorporated.

For standard silicon processes, the photodiode 184 detects a minimum of5 photons. However, to ensure reliable detection, the minimum can be setto 10 photons. Therefore with the quantum efficiency range being 0.3 to0.5 (as discussed above), the fluorescence emission from the probesshould be a minimum of 17 photons but 30 photons would incorporate asuitable margin of error for reliable detection.

Electrochemiluminescence as an Alternative Detection Method

Electrochemiluminescence (ECL) involves the generation of species atelectrode surfaces that then undergo electron-transfer reactions to formexcited states that emit light. Electrochemiluminescence differs fromnormal chemiluminescence in that formation of the excited species relieson oxidation or reduction of the luminophore or a coreactant at anelectrode. Coreactants, in this context, are additional reagents addedto the ECL solution which enhance the efficiency of ECL emission. Innormal chemiluminescence, the excited species form purely through mixingof suitable reagents. The emitting atom or complex is traditionallyreferred to as a luminophore. In brief, ECL relies on generating anexcited state of the luminophore, at which point a photon will beemitted. As with any such process, it is possible for an alternate pathto be taken from the excited state which does not lead to the desiredlight emission (i.e. quenching).

Embodiments of the test module that use ECL instead of fluorescencedetection do not require an excitation LED. Electrodes are fabricatedwithin the hybridization chambers to provide the electrical pulse forECL generation and the photons are detected using the photosensor 44.The duration and voltage of the electrical pulse are controlled; in someembodiments, control over the current is used as an alternative tocontrolling the voltage.

Luminophore and Quencher

The ruthenium complex, [Ru(bpy)_(3]) ²⁺, described previously for use asa fluorescent reporter in the probes, can also be used as a luminophorein an ECL reaction in the hybridization chambers, with TPrA(tri-n-propylamine (CH₃CH₂—CH₂)₃N) as the coreactant. Coreactant ECL hasthe benefit that luminophores are not consumed after photon emission andthe reagents are available for the process to repeat. Furthermore, the[Ru(bpy)₃]²⁺/TPrA ECL system provides good signal levels atphysiologically relevant conditions of pH in aqueous solutions.Alternative coreactants which can produce equivalent or better resultsthan TPrA with ruthenium complexes are N-butyldiethanolamine and2-(dibutylamino)ethanol.

FIG. 76 illustrates the reactions occurring during an ECL process inwhich [Ru(bpy)₃]²⁺ is the luminophore 864 and TPrA is the coreactant866. ECL emission 862 in the [Ru(bpy)₃]²⁺/TPrA ECL system follows theoxidation of both Ru(bpy)₃ ²⁺ and TPrA at the anode 860. The reactionsare as follows:

Ru(bpy)₃ ²⁺−e⁻→Ru(bpy)₃ ³⁺  (1)

TPrA−e⁻→[TPrA^(•)]⁺→TPrA^(•)+H⁺  (2)

Ru(bpy)₃ ³⁺+TPrA¹⁰⁸ Ru(bpy)₃ ^(•2+)+products   (3)

Ru(bpy)₃ ^(•2+)→Ru(bpy)₃ ²⁺+hυ  (4

The wavelength of the emitted light 862 is around 620 nm and the anodepotential is around 1.1 V with respect to a Ag/AgCl reference electrode.For the [Ru(bpy)_(3]) ²⁺/TPrA ECL system, either the Black HoleQuencher, BHQ 2, or Iowa Black RQ described previously, would be asuitable quencher. In the embodiments described here, the quencher is afunctional moiety which is initially attached to the probe, but otherembodiments are possible in which the quencher is a separate moleculefree in solution.

Hybridization Probes for ECL Detection

FIGS. 93 and 94 show the hybridization-responsive ECL probes 237. Theseare often referred to as molecular beacons and are stem-and-loop probes,generated from a single strand of nucleic acid, that luminesce uponhybridization to complementary nucleic acids. FIG. 93 shows a single ECLprobe 237 prior to hybridization with a target nucleic acid sequence238. The probe has a loop 240, stem 242, a luminophore 864 at the 5′end, and a quencher 248 at the 3′ end. The loop 240 consists of asequence complementary to the target nucleic acid sequence 238.Complementary sequences on either side of the probe sequence annealtogether to form the stem 242.

In the absence of a complementary target sequence, the probe remainsclosed as shown in FIG. 93. The stem 242 keeps the luminophore-quencherpair in close proximity to each other, such that significant resonantenergy transfer can occur between them, substantially eliminating theability of the luminophore to emit light after electrochemicalexcitation.

FIG. 94 shows the ECL probe 237 in an open or hybridized configuration.Upon hybridization to a complementary target nucleic acid sequence 238,the stem-and-loop structure is disrupted, the luminophore 864 andquencher 248 are spatially separated, thus restoring the ability of theluminophore 864 to emit light. The ECL emission 862 is opticallydetected as an indication that the probe has hybridized.

The probes hybridize with very high specificity with complementarytargets, since the stem helix of the probe is designed to be more stablethan a probe-target helix with a single nucleotide that is notcomplementary. Since double-stranded DNA is relatively rigid, it issterically impossible for the probe-target helix and the stem helix tocoexist.

Primer-Linked ECL Probes

Primer-linked stem-and-loop probes and primer-linked linear probes,otherwise known as scorpion probes, are an alternative to molecularbeacons and can be used for real-time and quantitative nucleic acidamplification in the LOC device. Real-time amplification is performeddirectly in the hybridization chambers of the LOC device. The benefit ofusing primer-linked probes is that the probe element is physicallylinked to the primer, thus only requiring a single hybridization eventto occur during the nucleic acid amplification rather than separatehybridizations of the primers and probes being required. This ensuresthat the reaction is effectively instantaneous and results in strongersignals, shorter reaction times and better discrimination than whenusing separate primers and probes. The probes (along with polymerase andthe amplification mix) would be deposited into the hybridizationchambers 180 during fabrication and there would be no need for anamplification section on the LOC device. Alternatively, theamplification section is left unused or used for other reactions.

Primer-Linked Linear ECL Probes

FIGS. 95 and 96 show a primer-linked linear ECL probe 693 during theinitial round of nucleic acid amplification and in its hybridizedconfiguration during subsequent rounds of nucleic acid amplification,respectively. Referring to FIG. 95, the primer-linked linear ECL probe693 has a double-stranded stem segment 242. One of the strandsincorporates the primer linked probe sequence 696 which is homologous toa region on the target nucleic acid 696 and is labelled on its 5′ endwith luminophore 864, and linked on its 3′ end to an oligonucleotideprimer 700 via an amplification blocker 694. The other strand of thestem 242 is labelled at its 3 end with a quencher molecule 248. Afterthe initial round of nucleic acid amplification has completed, the probecan loop around and hybridize to the extended strand with the, now,complementary sequence 698. During the initial round of nucleic acidamplification, the oligonucleotide primer 700 anneals to the target DNA238 (see FIG. 95) and is then extended, forming a DNA strand containingboth the probe sequence and the amplification product. The amplificationblocker 694 prevents the polymerase from reading through and copying theprobe region 696. Upon subsequent denaturation, the extendedoligonucleotide primer 700/template hybrid is dissociated and so is thedouble stranded stem 242 of the primer-linked linear probe, thusreleasing the quencher 248. Once the temperature decreases for theannealing and extension steps, the primer linked probe sequence 696 ofthe primer-linked linear ECL probe curls around and hybridizes to theamplified complementary sequence 698 on the extended strand and lightemission is detected indicating the presence of the target DNA.Non-extended primer-linked linear ECL probes retain theirdouble-stranded stem and light emission remains quenched. This detectionmethod is particularly well suited for fast detection systems as itrelies on a single-molecule process.

Primer-Linked Stem-and-Loop ECL Probes

FIGS. 97A to 97F show the operation of a primer-linked stem-and-loop ECLprobe 705. Referring to FIG. 97A, the primer-linked stem-and-loop ECLprobe 705 has a stem 242 of complementary double-stranded DNA and a loop240 which incorporates the probe sequence. One of the stem strands 708is labelled at its 5′ end with luminophore 864. The other strand 710 islabelled with a 3′-end quencher 248 and carries both the amplificationblocker 694 and oligonucleotide primer 700. During the initialdenaturation phase (see FIG. 97B), the strands of the target nucleicacid 238 separate, as does the stem 242 of the primer-linkedstem-and-loop ECL probe 705. When the temperature cools for theannealing phase (see FIG. 97C), the oligonucleotide primer 700 on theprimer-linked stem-and-loop ECL probe 705 hybridizes to the targetnucleic acid sequence 238. During extension (see FIG. 97D), thecomplement 706 to the target nucleic acid sequence 238 is synthesizedforming a DNA strand containing both the probe sequence 705 and theamplified product. The amplification blocker 694 prevents the polymerasefrom reading through and copying the probe region 705. When the probenext anneals, following denaturation (see FIG. 97E), the probe sequenceof the loop segment 240 of the primer-linked stem-and-loop probe (seeFIG. 97F) anneals to the complementary sequence 706 on the extendedstrand. This configuration leaves the luminophore 864 relatively remotefrom the quencher 248, resulting in a significant increase in lightemission.

ECL Control Probes

The hybridization chamber array 110 includes some hybridization chambers180 with positive and negative ECL control probes used for assay qualitycontrol. FIGS. 98 and 99 schematically illustrate negative control ECLprobes 786 without a luminophore, and FIGS. 100 and 101 are sketches ofpositive control ECL probes 787 without a quencher. The positive andnegative control ECL probes have a stem-and-loop structure like the ECLprobes described above. However, an ECL signal 862 (see FIG. 94) willalways be emitted from positive control ECL probes 787 and no ECL signal862 is ever emitted from negative control ECL probes 786, regardless ofwhether the probes hybridize into an open configuration or remainclosed.

Referring to FIGS. 98 and 99, the negative control ECL probe 786 has noluminophore (and may or may not have a quencher 248). Hence, whether thetarget nucleic acid sequence 238 hybridizes with the probe as shown inFIG. 99, or the probe remains in its stem 242 and loop 240 configurationas shown in FIG. 98, the ECL signal is negligible. Alternatively, thenegative control ECL probe could be designed so that it always remainsquenched. For example, by having an artificial probe (loop) sequence 240that will not hybridize to any nucleic acid sequence within the sampleunder investigation, the stem 242 of the probe molecule willre-hybridize to itself and the luminophore and quencher will remain inclose proximity and no appreciable ECL signal will be detected. Thisnegative control would account for any low level emission that may occurif the quenching is not complete.

Conversely, the positive control ECL probe 787 is constructed without aquencher as illustrated in FIGS. 100 and 101. Nothing quenches the ECLemission 862 from the luminophore 864 regardless of whether the positivecontrol probe 787 hybridizes with the target nucleic acid sequence 238.

FIGS. 91 and 92 show another possibility for constructing a positivecontrol chamber. In this case, the calibration chambers 382 which aresealed from the amplicon (or any flow containing target molecules) canbe filled with the ECL luminophore solution such that a positive signalis always detected at the electrode

Similarly, the control chambers can be negative control chambers becausethe lack of inlets prevents any targets from reaching the probes suchthat an ECL signal is never detected.

FIG. 52 shows a possible distribution of the positive and negativecontrol probes (378 and 380 respectively) throughout the hybridizationchamber array 110. For ECL, positive and negative control ECL probes 786and 787 would replace control fluorescent probes 378 and 380,respectively. The control probes are placed in hybridization chambers180 along a line extending diagonally across the hybridization chamberarray 110. However, the arrangement of the control probes within thearray is arbitrary (as is the configuration of the hybridization chamberarray 110).

Calibration Chambers for ECL Detection

The non-uniformity of the electrical characteristic of the photodiode184, response to any ambient light present at the sensor array, andlight originating at other locations in the array, introduce backgroundnoise and offset into the output signal. This background is removed fromeach output signal by calibration chambers 382 in the hybridizationchamber array 110 which either do not contain any probes, contain probesthat have no ECL luminophore, or contain probes with a luminophore andquencher configured such that quenching is always expected to occur. Thenumber and arrangement of the calibration chambers 382 throughout thehybridization chamber array is arbitrary. However, the calibration ismore accurate if photodiodes 184 are calibrated by a calibration chamber382 that is relatively proximate. Referring to FIG. 116, thehybridization chamber array 110 has one calibration chamber 382 forevery eight hybridization chambers 180. That is, a calibration chamber382 is positioned in the middle of every three by three square ofhybridization chambers 180. In this configuration, the hybridizationchambers 180 are calibrated by a calibration chamber 382 that isimmediately adjacent.

FIG. 75 shows a differential imager circuit 788 used to substract thesignal from the photodiode 184 corresponding to the calibration chamber382 as a result of the applied electrical pulse, from the ECL signalfrom the surrounding hybridization chambers 180. The differential imagercircuit 788 samples the signal from the pixel 790 and a “dummy” pixel792. Signals arising from ambient light in the region of the chamberarray are also subtracted. The signals from the pixel 790 are small(i.e. close to dark signal), and without a reference to a dark level itis hard to differentiate between the background and a very small signal.During use, the “read_row” 794 and “read_row_d” 795 are activated and M4797 an MD4 801 transistors are turned on. Switches 807 and 809 areclosed such that the outputs from the pixel 790 and “dummy” pixel 792are stored on pixel capacitor 803 and dummy pixel capacitor 805respectively. After the pixel signals have been stored, switches 807 and809 are deactivated. Then the “read_col” switch 811 and dummy “read_col”switch 813 are closed, and the switched capacitor amplifier 815 at theoutput amplifies the differential signal 817.

ECL Levels and Signal Efficiency

The normal metric of efficiency in ECL is the number of photons obtainedper “Faradaic” electron, i.e. per electron which participates in theelectrochemistry. The ECL efficiency is denoted φ_(ECL):

$\begin{matrix}{\varphi_{ECL} = \frac{\int_{0}^{t}{I{\tau}}}{\left( \frac{N_{A}}{F} \right){\int_{0}^{t}{i{\tau}}}}} & (5)\end{matrix}$

where I is the intensity in photons per second, i is the current inamperes, F is Faraday's constant, and N_(A) is Avogadro's constant.

Efficiency of Coreactant ECL

Annihilation ECL in deoxygenated, aprotic solutions (e.g.nitrogen-flushed acetonitrile solutions) is simple enough to allowefficiency measurements, and the consensus value of φ_(ECL) is around5%. Coreactant systems, however, have been generally declared to bebeyond meaningful direct measurements of efficiency. Instead, emissionintensity is related by scaling to easily-prepared standard solutionssuch as Ru(bpy)₃ ²⁺, measured in the same format. The literature (seefor example J. K. Leland and M. J. Powell, J. Electrochem. Soc., 137,3127 (1990), and R. Pyati and M. M. Richter, Annu. Rep. Prog. Chem. C,103, 12-78 (2007)) indicates that (without enhancers such assurfactants), the efficiency of Ru(bpy)₃ ²⁺ ECL with TPrA coreactantspeaks at levels comparable to the 5% seen for annihilation ECL inacetonitrile (e.g. 2% efficiency; see I. Rubinstein & A. J. Bard, J. Am.Chem. Soc., 103 512-516 (1981)).

ECL Potentials

The voltage at the working electrode for the Ru(bpy)₃ ²⁺/TPrA system isapproximately +1.1 V (generally measured in the literature with respectto a reference Ag/AgCl electrode). Voltages this high shorten electrodelifetimes but this is not an issue for single-use devices such as theLOC device used in the present diagnostic system.

The ideal voltage between the anode and cathode depends on thecombination of solution components and electrode materials. Selectingthe correct voltage can require compromising between the highest signallevels, reagent and electrode stability, and the activation of undesiredside reactions such as electrolysis of the water in the chamber. Intests on buffered aqueous Ru(bpy)₃]²⁺/coreactant solution and platinumelectrodes, the ECL emission is maximized at 2.1-2.2 V (depending on thecoreactant choice). Emission intensities drop to <75% of the peak valuesfor voltages below 1.9 V and above 2.6 V, and to <50% of the peak valuesfor voltages below 1.7 V and above 2.8 V. A preferred anode-cathodevoltage difference for ECL operation in such systems is therefore1.7-2.8 V, with the range 1.9-2.6 V being particularly preferred. Thisallows maximization of the emission intensity as a function of voltage,while avoiding voltages at which significant gas evolution at theelectrodes is observed.

ECL Emission Wavelength

The wavelength of the emitted light 862 from ECL has an intensity peakat around 620 nm (measured in air or vacuum), and the emission spans arelatively broad wavelength range. Significant emission occurs atwavelengths from around 550 nm to 700 nm. Furthermore, the peak emissionwavelength can vary by ˜10% due to changes in the chemical environmentaround the active species. The LOC device embodiments described here,which incorporate no wavelength-specific filters, have two advantagesfor capturing signals with such a broad and variable spectrum. The firstadvantage is sensitivity: any wavelength filter reduces lighttransmission, even within its pass band, so efficiency is improved bynot including a filter. The second advantage is flexibility: adjustmentof filter pass bands is not required after minor reagent changes, andthe signals are less dependent on minor differences in non-targetcomponents of the input sample.

Solution Volume Participating ECL

ECL relies on the availability of luminophore (and coreactant) insolution. However, as illustrated in FIG. 78, the excited species 868are generated only in the solution 872 near the electrodes 860 and 870.The parameter boundary layer depth in the models presented here, is thedepth of the layer of solution 872 around the electrode 860 in which theexcited species 868 are generated.

This is a simplification, since solution dynamics can drive theavailable concentration upward or downward:

-   -   Increased availability: diffusion and electrophoretic effects        will allow exchange with more of the solution.    -   Decreased availability: reagents can adsorb onto the electrodes        and may become unavailable to the ECL process.

For a boundary layer depth value of 0.5 μm, the following observationsare made:

ECL is observed in experiments where conjugation to magnetic beads withdiameters up to 4.5 μm is used to attract the luminophore 864 to theanode 860.

Ru(bpy)₃ ²⁺/TPrA ECL emission 862 as a function of electrode spacing,for interdigitated electrode arrays, was found to be maximised at a 0.8μm electrode spacing. The requirement for a coreactant 866 in aqueoussolutions 872 can be lifted when electrode spacings are ˜2 μm. Thisindicates that the excited species 868 diffuse multiple microns, whichimplies diffusive exchange on a similar scale for the species in theground state.

Steady State and Pulsed Operation

During pulsed activation of the electrodes 860 and 870, the intensity ofthe ECL emission 862 (see FIG. 94) is generally higher than theintensity of the emission 862 from steady-state activation of theelectrodes. Accordingly, the activation signal to the electrodes 860 and870 is pulse-width modulated (PWM) by the CMOS circuitry 86 (see FIG.83).

Reagent Recycling and Species Lifetime

The Ru complex is not consumed in the Ru(bpy)₃ ²⁺/TPrA ECL system, sothe intensity of emission 862 does not reduce with successive reactioncycles. The lifetime of the rate-limiting step is approximately 0.2milliseconds giving a total reaction recycling time of approximately 1millisecond.

Electrophoretic Effects and Other Constraints

Given the complexity of the solutions in the hybridization chamber, alarge number of phenomena take place when the ECL voltage is turned on.Electrophoresis of macromolecules, ohmic conduction, and capacitiveeffects from small ion migration occur simultaneously.

Electrophoresis of the oligonucleotides (probes and amplicon) cancomplicate the detection of probe-target hybrids, as DNA is highlynegatively charged and attracted to the anode 860. The time scale forthis motion is typically short (in the order of milliseconds).Electrophoretic effects are strong even though the voltages are moderate(˜1 V), because the separation between the anode 860 and cathode 870 issmall.

Electrophoresis enhances the ECL emission 862 in some embodiments of theLOC device and degrades the emission in others. This is addressed byincreasing or decreasing the electrode spacing to get the associatedincreases or decreases in electrophoretic effect. Interdigitation of theanode 860 and the cathode 870 above the photodiode 184 represents theextreme case of minimizing this separation. Such an arrangement producesECL, even in the absence of a coreactant 866 at carbon electrodes 860and 870.

Ohmic Heating (DC Current)

The current required to maintain an ECL voltage of ˜2.2 V, is determinedas follows with reference to the ECL cell 874 schematically illustratedin FIG. 79.

The DC current through the chamber is determined by two resistances: theinterface resistance R_(i) between the electrodes 860 and 870 and thebulk of the solution, and the solution resistance R_(s) which is derivedfrom the bulk solution resistivity and conduction path geometry. Forsolutions with ionic strengths relevant to the conditions in LOCdevices, the chamber resistance is dominated by interfacial resistancesat the electrodes 860 and 870, and R_(s) can be neglected.

The effect of the interfacial resistance is estimated by scalingmeasurements of macroscopic current flow through similar solutions forthe electrode geometries in the LOC devices.

Macroscopic measurements of current density through a similar solution,at platinum electrodes, were taken. Consistent with the worst-case (highcurrent) approach being taken, overall ionic strength and ECL reactantconcentrations in the test solution were higher than those used in theLOC devices. The anode area was smaller than the cathode area, and wassurrounded by a cathode with comparable area in a ring geometry. For ananode consisting of a circle 2 mm in diameter, the current measured was1.1 mA, giving a current density of 350 A/m².

In the heating model, the electrode area is for the square ring geometryschematically illustrated in FIG. 79. The anode is a ring with width 1μm and thickness 1 μm. The surface area is 196 square microns, andtherefore the calculated current I=69 nA. The heating (power=V²/R) wasmodelled for the worst case in which all the heat goes into raising thetemperature of the water in the chamber. This leads to heating ofchamber contents at 5.8° C./s, at a voltage difference of 2.2 V, if noallowance for heat removal by the bulk of the LOC device is made.

Heating of the chambers by ˜20° C. can cause denaturation of mosthybridization probes. For highly specific probes intended for mutationdetection, it is preferable to further restrict heating to 4° C. orless. With this level of temperature stability, single basemismatch-sensitive hybridization, using appropriately designedsequences, becomes feasible. This allows the detection of mutations andallelic differences at the level of single nucleotide polymorphisms.Hence the DC current is applied to the electrodes 860 and 870 for 0.69s, to limit the heating to 4° C.

A current of ˜69 nA passing through the chamber is far more than can beaccommodated as Faradaic current by the ECL species at micromolarconcentrations. Therefore, low-duty-cycle pulsing of the electrodes 860and 870 to further reduce heating (to 1° C. or less) while maintainingsufficient ECL emission 862, does not introduce complications associatedwith reagent depletion. In other embodiments, the current is reduced to0.1 nA which removes the need for pulsed activation of the electrodes.Even at currents as low as 0.1 nA, the ECL emission 862 isluminophore-limited.

Chamber and Electrode Geometry Maximizing Optical Coupling Between ECLLuminescence and Photosensor

The immediate chemical precursors of ECL luminescence are generatedwithin nanometres of the working electrode. Referring again to FIG. 78,light emission (the excited species 868) generally occurs within micronsor less of that location. Hence the volume immediately adjacent to theworking electrode (anode 860) is visible to the corresponding photodiode184 of the photosensor 44. Accordingly, the electrodes 860 and 870 aredirectly adjacent the active surface area 185 of the correspondingphotodiode 184 in the photosensor 44. Furthermore, the anode 860 isshaped to increase the length of its lateral periphery ‘seen’ by thephotodiode 184. This aims to maximize the volume of excited species 868that can be detected by the underlying photodiode 184.

FIG. 77 schematically illustrates three embodiments of the anode 860. Acomb structure anode 878 has the advantage that the parallel fingers 880can be interdigitated with the fingers of a cathode 870. Theinterdigitated configuration is shown in FIG. 84, and in a partial viewof a LOC layout in FIGS. 90 and 92. The interdigitated configurationprovides a uniform dielectric gap 876 (see FIG. 78) that is relativelynarrow (1 to 2 microns) and the interdigitated comb structure isrelatively simple for the lithographic fabrication process. As discussedabove, a relatively narrow dielectric gap 876 between the electrodes 860and 870 obviates the need for a coreactant in some solutions 872, as theexcited species 868 will diffuse between anode and cathode. The removalof the requirement for a coreactant removes the potential chemicalimpact of the coreactant on the various assay chemistries and provides awider range of possible assay options.

Referring again to FIG. 77, some embodiments of the anode 860 have aserpentine configuration 882. To achieve high periphery length whilemaintaining tolerance against fabrication errors, it is convenient toform wide, rectangular meanders 884.

The anode may have a more complex configuration 886 if necessary ordesirable. For example, it may have a crenulated section 888, a branchedstructure 890, or a combination of the two. Partial views of LOC designsincorporating a branched structure 890 are shown in FIGS. 115 and 116.The more complicated configurations such as 886 provide a long length oflateral periphery, and are best suited to solution chemistries where acoreactant is employed since patterning a closely-spaced opposingcathode is more difficult.

Electrode Thickness

Generally, ECL cells involve a planar working electrode which is viewedexternally. Also, traditional microfabrication techniques for metallayers tend to lead to planar structures with metal thicknesses ofapproximately 1 micron. As has been indicated earlier, and shownschematically in FIGS. 77, 80 and 81, increasing the length of lateralperiphery enhances the coupling between the ECL emission and thephotodiode 184.

A second strategy to further increase the efficiency of collection ofemitted light 862 (see FIG. 94) by the photodiode 184 is to increase thethickness of the anode 860. This is shown schematically in FIG. 78. Thepart of the participating volume 892 adjacent to the walls of theworking electrode is the region most efficiently coupled to thephotodiode 184. Therefore, for a given width of working electrode 860,the overall collection efficiency of the emitted light 862 can beimproved by increasing the thickness of the electrodes. Further, sincehigh current carrying capacity is not required, the width of the workingelectrode 860 is reduced as far as is practical. The thickness of theelectrodes 860 and 870 can not increase without restrictions. Notingthat the feature and separation sizes of the electrodes are likely to beof the order of 1 micron, and that liquid filling makes gaps which arewider than they are deep unfavourable, the optimum practical thicknessfor the electrodes is 0.25 micron to 2 microns.

Electrode Spacing

The spacing between the electrodes 860 and 870 is important for thequality of signals in LOC devices, particularly in embodiments where theelectrodes are interdigitated. In embodiments where the anode 860 is abranched structure such as shown in FIG. 77 and FIG. 81, the spacingbetween adjacent elements can also be important. ECL emissionefficiency, and the collection efficiency of the emitted light, shouldboth be maximised.

Generation of ECL emission tends to favour electrode spacings on theorder of one micron or less. Small spacings are particularly attractivewhen performing ECL in the absence of a coreactant. The fact that thespacing can be comparable to the wavelength of the emitted light 862 isof limited importance. Therefore, in many embodiments where the emittedlight 862 (see FIG. 94) is measured at a location which does not requirethat the light have passed between the electrodes 860 and 870, makingthe electrode spacing as small as practical is often the goal. Inembodiments where the emitted light 862 must pass between the electrodes860 and 870, however, it becomes necessary to move beyond consideringjust the ECL emission process, and consider the wave properties oflight.

The wavelength of the emitted light 862 from ECL of Ru(bpy)₃ ²⁺ isaround 620 nm, and therefore 460 nm (0.46 microns) in water. Inembodiments where the photodiode 184 and the ECL excited species 868 areon different sides of the electrode structure, and the electrodestructure is metallic, the emitted light 862 must pass through a gapbetween elements of the metallic structures. If this gap is comparableto the wavelength of the light, diffraction generally reduces theintensity of propagating light which reaches the photodiode 184. Incases where the emitted light 862 is incident on the gap at largeangles, however, evanescent mode coupling can be harnessed to improvethe strength of collected signals. Two measures are taken in the LOCdevices to enhance the efficiency of coupling between the photodiode 184and the emitted light 862.

First, the separation between metallic elements is not reduced belowapproximately the wavelength of the emitted light in water, i.e.approximately 0.4 microns. When combined with other observationsregarding small separations between interdigitated electrodes, thisindicates an optimal range for the electrode spacing of 0.4 to 2microns.

Second, the distance from the gap between elements to the photodiode 184is minimised. In the LOC device embodiments described here, thisindicates that the total thickness of layers between the electrodes 860and 870 and the photodiode 184 be one micron or less. In embodimentswhere multiple layers are present between the electrodes and thephotodiode, arranging their thicknesses to be quarter-wave orthree-quarter wave layers has the further benefit of suppressingreflection of the emitted light 862.

Electrode Models

FIG. 78 is a schematic partial cross-section of the electrodes 860 and870 in the hybridization chamber. The volume around the lateralperiphery of the anode 860 occupied by the excited species 868, issometimes referred to as the participating volume 892. The occludedregion 894 above the anode 860 is ignored because its optical couplingto the photodiode 184 is negligible.

A technique for determining whether a particular electrode configurationprovides a foundation for the level of ECL emission 862 for theunderlying photodiode 184 is set out below with reference to FIGS. 79,80 and 81.

FIG. 79 is a ring geometry in which the anode 860 is around the edge ofphotodiode 184. In FIG. 80, the anode 860 is positioned within theperiphery of the photodiode 184. FIG. 81 shows a more complexconfiguration in which the anode 860 has a series of parallel fingers880 to increase the length of its lateral edges.

For all of the above configurations, the model calculations are asfollows. For a participating volume 892 of solution V_(ECL), the totaleffective number of emitters N_(em) is:

N _(em) =N _(lum)·τ_(p)/τ_(ECL) =V _(ECL) C _(L) N _(A)·τ_(p) /τ _(ECL)  (6)

where the participating number of luminophoresN_(lum)=V_(ECL)C_(L)N_(A), τ_(ECL) is the lifetime of the ECL process,C_(L) is the luminophore concentration, τ_(p) is the pulse duration, andN_(A) is Avogadro's number.

The number of isotropically emitted photons N_(phot) is:

N_(phot)=φ_(ECL)N_(em)   (7)

where φ_(ECL) is the ECL efficiency, defined as the average number ofphotons emitted by the ECL reaction of a single luminophore.

The signal count of electrons, S, from the photodiode is then

S=N_(phot)·φ_(o)φ_(q),   (8)

where φ_(o) is the optical coupling efficiency (the number of photonsabsorbed by the photodiode 184) and φ_(q) is the photodiode quantumefficiency. The signal is therefore:

$\begin{matrix}{S = {V_{ECL}C_{L}N_{A}\frac{\tau_{p}}{\tau_{ECL}}\varphi_{ECL}\varphi_{o}\varphi_{q}}} & (9)\end{matrix}$

For FIGS. 79 and 80 electrode configurations, φ_(o) is:

φ_(o)=(25% photons which are directed towards the photodiode 184)×(10%of photons which are not reflected)

i.e., φ_(o)=2.5% for configurations shown in FIGS. 79 and 80

For the electrode configuration of FIG. 81, 50% of photons are emittedin a direction pointing towards the photodiode 184, but the absorptionefficiency as a function of angle is unchanged, so

φ_(o)=(50% photons which are directed towards the photodiode)×(10% ofphotons which are not reflected)

i.e., φ_(o)=5% for the configuration of FIG. 81.

The participating volume 892 depends on the electrode configuration, anddetails are presented in the corresponding sections.

The input parameters for the calculations are listed in the following:

TABLE 5 Input Parameters Parameter Value Comment Luminophore concen-2.89 μM Probe concentration tration C_(L) calculated previously ECLrecycling period 1 ms Combined lifetimes of (lifetime) τ_(ECL) reactionsteps for luminophore. Boundary layer 0.5 μm Effective volume (includingdepth D diffusion and electro- phoresis) of solution participating inECL Duration of current 0.69 s Chosen to limit ohmic application τ_(p)heating to 4° C. (as described previously) Chamber X dimension 28 μmChamber Y dimension 28 μm Chamber height Z 8 μm Photodiode X dimension16 μm Photodiode Y dimension 16 μm Electrode thickness (i.e., 1 μmexposed edge height) Electrode layer minimum 1 μm Process critical widthand gap dimension Electrode interfacial 350 A/m² For ohmic heatingcurrent density Solution volume 0.5 Ω · m For ohmic heating resistivityVoltage difference 2.2 V applied (working − counter electrode)

Ring Geometry Around Periphery of Photodiode

Referring to FIG. 79, the anode 860 is a ring around the edge of thephotodiode 184. In this configuration, the participating volume 892 is:

V _(ECL)=4×[(layer beside the electrode wall)+(quarter-cylinder abovethe electrode wall)]

Calculation results:

-   Photons generated from a 0.5 μm boundary layer: 3.1×10⁵-   Electron counts in photodiode: 2.3×10³

This signal is readily detectable by the underlying photodiode 184 ofthe LOC device photosensor 44.

Additional Fingers to Increase Edge Length

Referring to FIG. 81, parallel fingers 880 are added across the anode860. Only horizontal edges shown in figure contribute to theparticipating volume 892, to avoid double-counting the perpendicularedges. The participating volume 892 is then:

V _(ECL)=(8×2)×[(layer beside the electrode wall)+(quarter-cylinderabove the electrode wall)]

Calculation results for FIG. 81 configuration:

-   Photons generated from a 0.5 μm boundary layer: 1.1×10⁶-   Electron counts in photodiode 184: 8.0×10³

This signal is easily detectable in the photodiode 184.

Complete Overlay

This configuration shown in FIG. 82 and FIG. 83 is included as alimiting case of maximum surface area coupling. In practice, 90% orbetter coupling between the electrode surface area and the activesurface area 185 of the photodiode 184 achieves a nearly optimal result,and even coupling of 50% of the photodiode active surface area 185 tothe electrode surface area provides most of the benefit of the completeoverlay configuration. Complete overlay can be achieved in twoembodiments: first, as indicated schematically in FIG. 82, by employinga transparent anode 860, in a plane parallel with that of the photodiode184 and with an area matched to that of the photodiode, and arrangingthe anode in immediate proximity to the photodiode 184, such thatemitted light 862 passes through the anode and onto the photodiode. In asecond embodiment shown schematically in FIG. 83, the anode 860 is againparallel to and registered with the photodiode area, but the solution872 fills a void between the anode 860 and the photodiode 184. Forsignal modelling of a complete overlay configuration, the anode isassumed to be a complete layer above the photodiode 184, with half ofthe photons directed toward the photodiode 184 (absorption efficiencystill 10%).

-   Photons generated from a 0.5 μm boundary layer: 7.7×10⁵-   Electron counts in photodiode: 1.2×10⁴

It is possible to improve the signal and assay beyond the above modelsby using surfactants and probe immobilization at the anode.

Maximum Spacing Between ECL Probes and Photodiode

The on-chip detection of hybridization avoids the needs for detectionvia confocal microscopy (see Background of the Invention). Thisdeparture from traditional detection techniques is a significant factorin the time and cost savings associated with this system. Traditionaldetection requires imaging optics which necessarily uses lenses orcurved mirrors. By adopting non-imaging optics, the diagnostic systemavoids the need for a complex and bulky optical train. Positioning thephotodiode very close to the probes has the advantage of extremely highcollection efficiency: when the thickness of the material between theprobes and the photodiode is on the order of 1 micron, the angle ofcollection of emission light is up to 174°. This angle is calculated byconsidering light emitted from a probe at the centroid of the face ofthe hybridization chamber closest to the photodiode, which has a planaractive surface parallel to that chamber face. The cone of emissionangles within which light is able to be absorbed by the photodiode isdefined as having the emitting probe at its vertex and the corner of thesensor on the perimeter of its planar face. For a 16 micron×16 micronsensor, the vertex angle of this cone is 170°; in the limiting casewhere the photodiode is expanded so that its area matches that of the 28micron×26.5 micron hybridization chamber, the vertex angle is 174°. Aseparation between the chamber face and the photodiode active surface of1 micron or less is readily achievable.

Employing a non-imaging optics scheme does require the photodiode 184 tobe very close to the hybridization chamber in order to collectsufficient photons of fluorescence emission. The maximum spacing betweenthe photodiode and probes is determined as follows.

Utilizing a ruthenium chelate luminophore and the electrodeconfiguration of FIG. 81, we calculated 27,000 photons being absorbed byour 16 micron×16 micron sensor from the respective hybridizationchamber, to generate 8000 electrons assuming a sensor quantum efficiencyof 30%. In performing this calculation we assumed that thelight-collecting region of our hybridization chamber has a base areawhich is the same as our sensor area, one quarter of the total number ofthe hybridization photons is angled so as to reach the sensor, and aconservative 10% estimate for the proportion of photons which do notscatter away from the sensor-dielectric interface. That is, the lightgathering efficiency of the optical system is φ₀=0.025.

More accurately we can write φ₀=[(base area of the light-collectingregion of the hybridization chamber)/(photodetector area)][Ω/4π][10%absorbed], where Ω=solid angle subtended by the photodetector at arepresentative point on the base of the hybridization chamber. For aright square pyramid geometry:

Ω=4 arcsin(α²/(4d ₀ ²+α²)), where d₀=distance between the chamber andthe photodiode, and α is the photodiode dimension.

Each hybridization chamber releases 1.1×10⁶ photons. The selectedphotodetector has a detection threshold of 17 photons, and for values ofd₀ greater than ten times the sensor size (i.e., essentially normalincidence) the proportion of photons not reflected at the sensor surfacecan be increased from 10% to 90%. Therefore, the minimum opticalefficiency required is:

φ₀=17/(1.1×10⁶×0.9)=1.72×10⁻⁵

The base area of the light-emitting region of the hybridization chamber180 is 29 micron×19.75 micron.

Solving for d₀, we will get the maximum limiting distance between thebottom of our hybridization chamber and our photodetector to be d₀=1600microns. In this limit, the collection cone angle as defined above isonly 0.8°. It should be noted this analysis ignores the negligibleeffect of refraction.

LOC Variants

The LOC device 301 described and illustrated above in full is just oneof many possible LOC device designs. Variations of the LOC device thatuse different combinations of the various functional sections describedabove will now be described and/or shown as schematic flow-charts, fromsample inlet to detection, to illustrate some of the combinationspossible. The flow-charts have been divided, where appropriate, intosample input and preparation stage 288, extraction stage 290, incubationstage 291, amplification stage 292, pre-hybridization stage 293 anddetection stage 294. For all the LOC variants that are briefly describedor shown only in schematic form, the accompanying full layouts are notshown for reasons of clarity and succinctness. Also in the interests ofclarity, smaller functional units such as liquid sensors and temperaturesensors are not shown but it will be appreciated that these have beenincorporated into the appropriate locations in each of the following LOCdevice designs.

Microfluidic Device with Dialysis Device, LOC Device and InterconnectingCap

A microfluidic device 783 with dialysis device 784, LOC device 785 andinterconnecting cap 51 provides for greater modularity and improvedsensitivity. In contrast to LOC designs which combine all the functions(see, for example, LOC variant 729 in FIG. 89), separating the dialysisfunction from the LOC device allows different specialized dialysisdevices 784 to be developed that select for different targets. Thesespecialized dialysis devices 784 can be combined with a LOC device 785and interconnecting cap 51 to form a complete assay system. Furthermore,different LOC devices 785 optimized for different assay approaches canalso be developed and coordinated with different dialysis devices 784,thus providing an extremely powerful and flexible approach to systemdevelopment. It is also possible to deploy the LOC device without adialysis device for certain applications, or to combine multiple LOCdevices 785.

Each surface-micromachined chip constituent of the microfluidic device783 can be fabricated with the optimal and most cost effectivefabrication process. For example, the dialysis device 784 requires noCMOS circuitry and so can be manufactured utilizing less expensivematerials and fewer process steps. Furthermore, a larger and optimizeddialysis device 784 provides increased sensitivity, signal-to-noiseratio, and dynamic range for the assay system.

As diagrammatically shown in FIG. 102, the microfluidic device 783prepares the sample 288, and then extracts 290, incubates 291, amplifies292, and detects 294 pathogenic DNA using twelve separate amplificationchambers (112.1 to 112.12). The assembly employs multiple amplificationchambers to increase assay sensitivity and improve the signal-to-noiseratio. This LOC device utilizes ECL in the hybridization chamber arrays110.1 to 110.12 to detect probe-target hybrids. The system modularityprovides for different dialysis devices 784 to be employed to detectother targets, for example, leukocytes, erythrocytes, pathogens, ormolecules like free proteins or DNA within a liquid sample, and althoughthe current description describes pathogenic DNA detection, the skilledworker will appreciate that the microfluidic device 783 is not limitedto detecting this one target only.

FIG. 103 shows the microfluidic device 783 with dialysis device 784, LOCdevice 785, and interconnecting cap 51. The interconnecting cap 51consists of the reservoir layer 78, the cap channel layer 80, and theinterface layer 594. The interface layer 594 is positioned between thecap channel layer 80 and the MST channel layer 100 of the CMOS+MSTdevice 48. The interface layer 594 allows a more complex fluidicinterconnection between the reagent reservoirs and the MST layer 87without increasing the size of the silicon substrate 84. FIG. 104superimposes the reservoirs, the top channels, and the interfacechannels to illustrate the more sophisticated plumbing achieved with theinterface layer 594.

Referring to FIGS. 104 and 105, the sample (for example, blood) entersthe sample inlet 68 and capillary action draws it along the cap channel94 to the anticoagulant surface tension valve 118. As best shown in FIG.105, the anticoagulant surface tension valve 118 has two interfacechannels 596 and 598 in the interface layer 594. A reservoir-sideinterface channel 596 connects the reservoir outlet with the downtakes92 and a sample-side interface channel 598 connects the uptakes 96 withthe cap channel 94. Anticoagulant from the reservoir 54 flows throughthe MST channels 90 via the reservoir-side interface channel 596 untilthe meniscus is pinned at the uptakes 96. The sample flow along the capchannel 94 dips into the sample-side interface channel 598 to remove themeniscus so that the anticoagulant combines with the blood sample as itcontinues on to the pathogen dialysis section 70.

Referring to FIGS. 104 and 105, the pathogen dialysis section 70 in thisembodiment comprises an interface target channel 602 and an interfacewaste channel 604 fluidically coupled by a plurality of apertures of apredetermined threshold size. The aperture at the very upstream of thedialysis section 70 is different from the apertures downstream of it;the downstream apertures are holes with diameters of less than 8.0microns selected to allow targets to pass through to the interfacetarget channel 602. In the present embodiment the apertures are 3.0micron diameter holes 164 selected to allow pathogens to pass through tothe interface target channel 602. The pathogen dialysis section 70 isconfigured such that the sample flows through the channels and aperturesunder capillary action.

Referring to FIGS. 104 and 105, the blood sample flows through the capchannel 94 to the upstream end of the interface waste cell channel 604.The interface waste cell channel 604 is open to the 3.0 micron diameterapertures 164 leading to the dialysis MST channels 204. Each of thedialysis MST channels 204 lead from the 3.0 micron diameter apertures164 to respective dialysis uptake holes 168. The dialysis uptake holes168 are open to the interface target channel 602. However the uptakesare configured to pin a meniscus rather than allow capillary driven flowto continue.

The pathogen dialysis section 70 incorporates a bypass channel 600 forfilling the flow channel structures without trapped air bubbles. Thebypass channel 600 at the very upstream end of the pathogen dialysissection 70, has a CIF (capillary initiation feature) 202 to promotecapillary driven flow from the bypass channel 600 into the interfacetarget channel 602 (see FIGS. 104 and 105). The bypass channel also hasa wide meander to lengthen the flow-path from the interface waste cellchannel 604 to the interface target channel 602. The longer flow-pathdelays the sample flow such that it fills the interface target channel602 after the meniscus forms at the most upstream dialysis MST channel204. The sample flow starts at the upstream end and unpins the meniscusat each of the dialysis uptake holes 168 as the flow moves downstreamalong the interface waste channel 602. This ensures all the dialysisbottom channels fill with sample flow as the dialysis section fills.Without the bypass channel 600, or dialysis uptakes 168 configured topin a meniscus, some dialysis MST channels 204 may not fill. Similarly,an air bubble may form in the interface target channel 602. In eithercase, flow through the dialysis section can be substantially throttled.

The interface waste channel 604 feeds into the waste channel 72 and theinterface target channel 602 feeds into the target channel 74 (see FIGS.104 and 105). Five dialysis sections are joined in series by capchannels 72 and 74 to increase the efficiency of the dialysis process.At the exit of the fifth dialysis section, the sample flow with thetarget is drawn along the target channel 74 in the cap channel layeraway from the dialysis device 784 and into the LOC device 785 bycapillary driven flow. The waste channel 72 flows to the waste reservoir76 (see FIG. 103).

The LOC device 785 can also be employed as a standalone microfluidicdevice with an alternate cap suitable for a single device, and in thisconfiguration optional anticoagulant reservoir 55 can be used to supplyanticoagulant. One example of this standalone use of the LOC device 785is its use for whole blood analysis. Referring to FIGS. 106 and 107, thetarget flows into LOC device 785 along cap channel 74. It passes viaoptional anticoagulant surface tension valve 117, which is used to addthe contents of optional anticoagulant reservoir 55, and continues onuntil it reaches the lysis surface tension valve 128. In theconfiguration being described here, this optional reservoir and surfacetension valves are unused and do not affect the sample flow or operationof the LOC device.

As with the anticoagulant surface tension valve 118 described above, thelysis surface tension valve 128 has a lysis reservoir-side interfacechannel 606 and a lysis sample-side interface channel 608 (see FIG.107). Lysis reagent flows from the reservoir 56 to the lysisreservoir-side interface channel 606 via a cap channel 94. The reagentflows into the downtakes 92, through the MST channels 90 to the uptakes96 where the reagents pin a meniscus (see FIG. 107). Sample flow fromthe target channel 74 fills the lysis sample-side interface channel 608.The sample flow removes the menisci at the uptakes 96 and the lysisreagent combines with the sample as it flows into the chemical lysissection 130.

In the chemical lysis chamber section 130, the lysis reagent diffusivelymixes through the flow to lyse the target cells and release the geneticmaterial therein. The sample flow stops at the mixing section exit valve206. The mixing section exit valve is a boiling-initiated valve 206. Theliquid sensor 174 upstream of the valve provides feedback that thesample flow is about to reach the valve 206. If the CMOS circuitry 86 isprogrammed with a delay to ensure the target cells are completely lysed,the liquid sensor feedback initiates the delay period. After any delayperiod, the boiling-initiated valve 206 is activated and the downstreamliquid sensor 174 registers that the flow has resumed along the MSTchannel 90.

The lysed sample flow continues to the restriction enzyme, ligase andlinker surface tension valve 132. The operation of the surface tensionvalve 132 is the same as described earlier for the anticoagulant surfacetension valve 118. Referring to FIG. 107, when the lysed sample flowreaches the pinned-menisci at the surface tension valve 132, restrictionenzymes, ligase and linker primers are released from the reservoir 58and combine with the sample flow. The sample then flows through MSTchannel 90 to the heated microchannels of the incubation section 114.Referring to FIGS. 107 and 108, the incubation section 114 is composedof a serpentine microchannel 210 heated by heaters 154.

Referring to FIG. 108, the sample flow is stopped at the incubatoroutlet valve 207 until sufficient time has passed. The incubator outletvalve 207 is a boiling-initiated valve similar to the mixing sectionoutlet valve 206. The liquid sensor 174 at the beginning of theincubation section, in conjunction with the flow rate sensor 740 (seeFIG. 112) and CMOS circuitry 86, initiates an incubation time delay.After sufficient incubation, the incubator outlet valve 207 activatesand flow resumes flowing along the MST incubation exit channel 630 tothe polymerase surface tension valve 140 (see FIG. 109). Polymerase fromthe reservoir 62 combines with the sample flow as it travels theserpentine path of the amplification input channel 632.

Referring to FIGS. 108 and 109, the amplification input channel 632directs the sample flow past the twelve amplification mix surfacetension valves 138. Amplification mix in each of the amplification mixreservoirs 60.1-60.12 (see FIG. 109) flows through respective capchannels 94 to pin menisci at the amplification mix surface tensionvalves 138. The sample flow opens each of the surface tension valves inturn, and the amplification mix from the respective amplification mixreservoirs 60.1-60.12 entrains with the sample flow into each of thetwelve amplification chambers 112.1-112.12. The LOC device has CMOScircuitry that allows operative control of amplification section viatemperature sensors and heaters.

Referring to FIG. 110, each of the twelve amplification chambers112.1-112.12 has one of the amplification outlet valves 207,respectively. The amplification outlet valves 207 are boiling-initiatedvalves like the incubator outlet valve 207. The sample flow stops ateach amplification outlet valve 207. After amplification, theamplification outlet valves 207 open for the amplicon to flow into thehybridization chamber arrays 110.1-110.12 containing probes configuredto form probe-target hybrids with the target nucleic acid sequences, inthis case pathogenic DNA. The sample is drawn along the flow-path 176through each of the separate arrays 110.1-110.12 and into the individualhybridization chambers 180 via respective diffusion barrier inlets 175(see FIG. 90 and FIG. 111).

Referring to FIGS. 91 and 111, when the sample flow reaches theend-point liquid sensor 178, the hybridization heaters 182 are activatedafter a time delay to enhance the generation of probe-target hybrids.The flow rate sensor 740 (see FIG. 112) is included in the pathogenincubation section 114 to determine the time delay. After a suitabledelay for hybridization, an excitation current applied to the ECLelectrodes 860 and 870 (see FIGS. 111 and 114) causes the probe-targethybrids to emit photons of light that are detected with the photosensor44 in the underlying CMOS circuitry 86. The photosensor is comprised ofan array of photodiodes 184 positioned adjacent each of thehybridization chambers.

FIGS. 113 and 114 show the calibration chambers 382. They are used tocalibrate the photodiodes 184 to adjust for system noise and backgroundlevels as described elsewhere in this specification. Also, positive ECLcontrol probes 787 and negative ECL control probes 786 are placed insome of the hybridization chambers 180 for assay quality control. Avariant of the calibration chambers 382 with interdigitated ECLelectrodes is shown in FIG. 92.

A humidifier 196 and humidity sensor 232 are used to control evaporationand condensation in the LOC device 785, particularly the hybridizationchamber array 110. FIG. 110 shows the major components of the humidifier196, the water reservoir 188 and evaporators 190.

Referring to FIG. 109, the evaporation-based telltale 189 indicateswhether the packaging of the device has been damaged during storage andthus whether the integrity and reliability of the microfluidic devicehas been compromised. During manufacture, a small drop of liquid isapplied to the liquid sensor 174 at the centre of the evaporation-basedtelltale 189. If the package seal is breached during storage then thedrop of liquid will evaporate. The presence or absence of the liquid canbe detected with the liquid sensor 174 and thus indicate the integrityof the seals on the microfluidic device.

CONCLUSION

The devices, systems and methods described here facilitate moleculardiagnostic tests at low cost with high speed and at the point-of-care.

The system and its components described above are purely illustrativeand the skilled worker in this field will readily recognize manyvariations and modifications which do not depart from the spirit andscope of the broad inventive concept.

1. A test module for analyzing a sample fluid containing targetmolecules, the test module comprising: an outer casing with a receptaclefor receiving the sample fluid; and, a microfluidic device having: adialysis device in fluid communication with the receptacle andconfigured to separate the target molecules from other constituents ofthe sample; a lab-on-a-chip (LOC) device for analyzing the targetmolecules; and, a cap overlaying the LOC device and the dialysis devicefor establishing fluid communication between the LOC device and thedialysis device.
 2. The test module according to claim 1 wherein thefluid sample is a sample of biological material including cells ofdifferent sizes, and the dialysis device has at least two channels witha plurality of apertures fluidically connecting the channels, theplurality of apertures being sized to correspond to a predeterminedthreshold size of the cells in the fluid sample.
 3. The test moduleaccording to claim 2 wherein the at least two channels and the pluralityof apertures are configured such that the sample flows though thechannels and the apertures under capillary action.
 4. The test moduleaccording to claim 3 wherein the at least two channels includes a targetchannel and a waste channel, the target channel being connected to thecap for capillary driven flow to the LOC device.
 5. The test moduleaccording to claim 4 wherein the target molecules are target nucleicacid sequences within cells in the sample fluid, and the LOC device hasa nucleic acid amplification section for amplifying the target nucleicacid sequences.
 6. The test module according to claim 5 wherein thetarget nucleic acid sequences are within the cells smaller than thepredetermined threshold.
 7. The test module according to claim 5 whereinthe LOC device has a hybridization section with an array of probes forhybridization with the target nucleic acid sequences to formprobe-target hybrids.
 8. The test module according to claim 7 whereinthe probes are configured to form probe-target hybrids with the targetnucleic acid sequences, the probe-target hybrids being configured toemit photons of light in response to an excitation electric current. 9.The test module according to claim 8 wherein the LOC device has CMOScircuitry for operative control of the PCR section, the CMOS circuitryhaving a photosensor for sensing photons emitted by the probe-targethybrids.
 10. The test module according to claim 9 wherein thehybridization section has an array of hybridization chambers containingthe probes for hybridization with the target nucleic acid sequences. 11.The test module according to claim 10 wherein the photosensor is anarray of photodiodes positioned adjacent each of the hybridizationchambers respectively.
 12. The test module according to claim 11 whereinthe CMOS circuitry has a digital memory for storing data relating to theprocessing of the fluid, the data including the probe details andlocation of each of the probes in the array of hybridization chambers.13. The test module according to claim 12 wherein the CMOS circuitry hasat least one temperature sensor for sensing the temperature at the arrayof hybridization chambers.
 14. The test module according to claim 13wherein the LOC device has a heater controlled by the CMOS circuitryusing feedback from the temperature sensor for maintaining the probesand the target nucleic acid sequences at a hybridization temperature.15. The test module according to claim 14 wherein the photodiodes areless than 1600 microns from the corresponding hybridization chamber. 16.The test module according to claim 15 wherein the probes have anelectrochemiluminescent (ECL) luminophore that emits a photon when in anexcited state.
 17. The test module of claim 16 wherein the hybridizationchambers have electrodes for exciting the ECL luminophores withelectrical current.
 18. The test module according to claim 17 whereinthe ECL probes each have a luminophore and a quencher positionedproximate the luminophore for quenching photons emitted by theluminophore such that hybridization with one of the target nucleic acidsequences moves the quencher away from the luminophore such that thephotons are not quenched.
 19. The test module according to claim 18wherein the CMOS circuitry has bond-pads for electrical connection to anexternal device, and is configured to convert output from thephotodiodes into a signal indicative of the ECL probes that hybridizedwith the target nucleic acid sequences, and provide the signal to thebond-pads for transmission to the external device.
 20. The test moduleaccording to claim 13 wherein the cap has at least one channel for fluidcommunication between the dialysis device and the LOC device, and aplurality of reservoirs for holding liquid reagents for addition to thesample.